This work is supported by the intramural fund of NIAID, NIH.

1. Culture and Differentiation of Human Neutrophil-like HL60 Cells
<ol>
	<li>Prepare RPMI (Roswell Park Memorial Institute) 1640 culture medium which contains RPMI 1640 medium, 10 % (v/v) fetal bovine serum (FBS), 0.1 mM sodium pyruvate, and 25 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid). Warm the RPMI 1640 culture medium to 37 &#176;C.</li>
	<li>Using a hemocytometer determine the cell density of HL60 cells to make sure the cells are in the log-phase growth stage. 
	NOTE: Cells are usually in log phase on the third day after passaging. The density of log-phase cells is usually 6-8 x 105 cells/ml. The cell density required to start a new culture is about 1.5 x 105 cells/ml. 
	NOTE: For example, for a 10 ml new culture in a T-75 flat flask, if the cell density of log-phase cells is 6 x 105 cells/ml, then 2.5 ml of log-phase cells (6 x 105) is diluted to 1.5 x 105 cells/ml in 10 ml new culture. To make a final volume of 10 ml culture, 7.5 ml of RPMI 1640 culture medium is added.</li>
	<li>Place the calculated volume of warm RPMI 1640 culture medium into a flat flask. For a T-75 flat flask culture, the total volume of the cell culture is 10 ml.</li>
	<li>Add the calculated volume of log-phase growing HL60 cells into the flat flask to reach a cell density of 1.5 x 105 cells/ml.</li>
	<li>Incubate the cells at 37 &#176;C in a humidified incubator with 5% CO2. 
	NOTE: The doubling time of HL60 is almost 24 hr.</li>
	<li>Passage the cells with RPMI 1640 culture medium every 2-3 days. It is important that the cell density of the HL60 cells never exceeds 1.5 x 106 cells/ml and the cell passage lasts no longer than 2 months.</li>
	<li>Differentiate the HL60 cells 5 days before the experiments. 
	NOTE: RPMI 1640 differentiation medium contains RPMI 1640 medium, 10% (v/v) fetal bovine serum (FBS), 0.1 mM sodium pyruvate, 25 mM HEPES, and 1.3% dimethyl sulfoxide (DMSO). In other words, it is 1.3% DMSO in RPMI 1640 culture medium.
	<ol>
		<li>To differentiate HL60 cells, pre-warm the RPMI 1640 culture medium to 37 &#176;C. Add the warm RPMI 1640 culture medium into a flat flask.</li>
		<li>For a final 10 ml HL60 differentiation culture, add 130 ul DMSO directly to the RPMI 1640 culture medium in the flat flask while swirling the flask so that the DMSO is rapidly diluted by the RPMI 1640 culture medium.</li>
		<li>Add log-phase HL60 cells to a final cell density of 1.5 x 105 cells/ml in a final 10 ml RPMI 1640 differentiation medium. Culture the HL60 cells in RPMI 1640 differentiation medium at 37 &#176;C in a humidified incubator with 5 % CO2 for 5 days.</li>
	</ol>
	</li>
</ol>
2. Coating the Cover Glass Surface of the 4well Chamber
<ol>
	<li>Take the bottle of 2% gelatin stock solution from the refrigerator, clean it with 70% ethanol, and place it in the hood. Take 1 ml 2% gelatin stock solution and return the remaining gelatin stock solution to the refrigerator immediately.</li>
	<li>Allow the gelatin solution to completely liquefy at 37 &#176;C and pipette it thoroughly. Dilute the 2% gelatin stock solution with 9 ml pre-warmed 1x Hanks&#39; balanced salt solution (HBSS) buffer to a final concentration of 0.2% gelatin.</li>
	<li>Add 0.5 ml or 2 ml 0.2% gelatin in HBSS to the two middle wells of a 4-well chamber or a 1-well chamber with a cover glass in the bottom, respectively. Tilt the plates in several directions so that the liquid covers the entire surface area.</li>
	<li>Place the plates in a 37 &#176;C incubator. The plates will be ready for use in 1 hr. They can be used for up to 5 days. Just before seeding cells, remove the gelatin solution from the 4-well or 1-well chambers.</li>
</ol>
3. Chemotaxis Assay Using Cell Mobility Analysis Device
<ol>
	<li>Prepare and warm RPMI 1640 starving medium, which is RPMI medium containing 0.1 mM sodium pyruvate (optional) and 25 mM HEPES.</li>
	<li>Prepare 100 &#956;l of 100 nM fMLP (N-formylmethionyl-leucyl-phenylalanine) in RPMI 1640 starving medium.</li>
	<li>Prepare 3 ml of 1% bovine serum albumin (BSA)/RPMI 1640 medium which contains 1% BSA in RPMI 1640 starving medium and 10 ml of 0.1% BSA/RPMI 1640 medium.</li>
	<li>Coat a 22 mm square coverslip and a 5 &#956;m cell mobility analysis device chip with freshly prepared 1% BSA/RPMI 1640 medium for 1 hr at RT.</li>
	<li>Assemble the Holder Using the Following Procedure:
	<ol>
		<li>Position the holder base with the levers in the vertical position so that the levers can tilt toward the user. Apply 70% ethanol solution to the surface of the 41 mm glass. Carefully wipe the surfaces with a wipe, taking care not to scratch them.</li>
		<li>Insert the 41 mm glass into the holder base. Put the BSA-coated 22 mm square coverslip on top of the 41 mm glass in the holder base. 
		NOTE: The coated square coverslip between the 41 mm glass and the chip allows chemotaxis to occur on the substratum of the coating surface.</li>
		<li>Insert the O-ring (small) into the bottom of the wafer housing, and mount the wafer housing into the holder base with the elliptical hole at the rear. Pull the inner level of the holder base forward to the horizontal position to lock the wafer housing in position.</li>
		<li>Blow any dust from the interior of the wafer housing with an air duster. Add 4 ml of 0.1% BSA/RPMI 1640 medium into the wafer housing.</li>
		<li>Mount the BSA-coated cell mobility analysis device chip in the center of the wafer housing with the structural face downwards in contact with the glass. 
		NOTE: That the chip needs to be inserted with the positioning mark (the hole in the top of the chip) at the rear.</li>
		<li>Affix the rubber gasket to the bottom of the wafer clamp by fitting the two protrusions on the rubber gasket into the holes.</li>
		<li>Mount the O-ring (large) onto the top of the wafer housing. Mount the wafer clamp with the sensor hole at the rear. Pull the outer lever of the housing base forward to the horizontal position to lock the wafer clamp in place.</li>
		<li>After assembling the holder, place the bottom of the holder on the light box to confirm that there are no air bubbles in the wells. If air bubbles are detected, remove them using the provided plastic syringe fitted with a sample loading tip.</li>
		<li>Remove the cover and put the assembly onto the plate on top of the cell mobility analysis device unit. Attach the sensor block to the holder.</li>
	</ol>
	</li>
	<li>Prepare HL60 Cells for Chemotaxis Assay:
	<ol>
		<li>Calculate the cell density of the differentiated HL60 cells with a hemocytometer. After 5 days of differentiation, the cell density is around 1.0 x 106 cells/ml.</li>
		<li>Calculate the final volume of HL60 cells at a final cell density of 2 x 106 cells/ml. For example, if the cell density of the differentiated HL60 cells is 1.0 x 106 cells/ml, then 10 ml of differentiated HL60 cells will be resuspended with 5 ml of 0.1% BSA/RPMI 1640 medium to reach 2 x 106 cells/ml.</li>
		<li>Centrifuge differentiated HL60 cells at 200 x g for 5 min at RT. Remove the supernatant and resuspend the cells with the calculated volume of 0.1% BSA/RPMI 1640 medium at step 3.6.2) to a final cell density of 2 x 106 cells/ml.</li>
	</ol>
	</li>
	<li>Start up the cell mobility analysis device unit and PC for image acquisition. Connect the unit to the PC. Turn on the cell mobility analysis device. Turn on the PC.</li>
	<li>Start Up the Cell Mobility Analysis Device Software.
	<ol>
		<li>Observe 5 control panels: camera, heater, shooting, memo, and camera images.</li>
		<li>On the camera image control panel, use &#34;Horizontal line&#34; or &#34;Vertical line&#34; to adjust the holder/view position in real time to center the camera in the camera image panel.</li>
		<li>On the camera control panel, select CH1 to CH6 to move the camera to the defined channel. To center the view fields of a channel, click &#34;Move L&#34; or &#34;Move R&#34; to adjust the X-coordinate of the camera and center the camera&#39;s position horizontally.</li>
		<li>Adjust the Y-coordinate of the camera by turning the position knob on the front panel of the cell mobility analysis device unit to vertically adjust the image to center the screen.</li>
		<li>On the heater control panel, set the &#34;holder temp&#34; to 37.0 &#176;C and the &#34;plate temp&#34; to 39 &#176;C. Click &#34;Heat&#34; to start heating. Also click &#34;holder&#34; to control the temperature using the thermal sensor connected to the holder.</li>
		<li>On the shooting panel, enter &#34;15 sec&#34; at &#34;Interval&#34; and &#34;30 min&#34; at &#34;Time&#34; for the intervals and the duration of the chemotaxis assay. Check &#34;Heater off at the end of shooting&#34; for safety reasons. Designate a destination for the stored images.</li>
		<li>On the memo panel, input the specifics of the experiments, such as cell line used, whether or not treatment was applied, and the concentration of chemo-attractants applied.</li>
		<li>Confirm the center position of all of the channels that will be used in the experiments, and repeat the adjustment for all channels if needed.</li>
	</ol>
	</li>
	<li>Inject and Align Cells as Follows:
	<ol>
		<li>Remove the buffer from the holder, and take out 8 &#956;l of buffer from the third well from the top for each channel.</li>
		<li>Use a syringe to inject 2 &#181;l of cells into the second well in the same channel while monitoring the screen in real time to control cell number and flow during injection. After the cells are nicely aligned, immediately add the 8&#160;&#181;l of buffer back. Repeat the same procedure for the other channels (Figure 1B).</li>
	</ol>
	</li>
	<li>Add 2 ml buffer back to the holder, and add 1 &#956;l 100 nM fMLP to the third well from the top. Start image acquisition and save the data. Analyze the data obtained using image tracing software9 (Figure 2).</li>
</ol>
4. Transfection with Electroporation
<ol>
	<li>Differentiate HL60 cells 5 days before the experiment as described in Section 1. Coat 4-well or 1-well chambers as instructed in Section 2.</li>
	<li>Prepare and warm RPMI 1640 electroporation recovery medium, which contains RPMI 1640 medium, 20% (v/v) FBS, 0.1 mM sodium pyruvate, and 25 mM HEPES, to 37 &#176;C.</li>
	<li>Right before starting the transfection experiment, add either 300 &#956;l or 3 ml RPMI 1640 culture medium into the wells of the pre-coated 4-well or 1-well chambers, respectively.</li>
	<li>Incubate the chambers in a humidified 37 &#176;C incubator with 5 % CO2. Use these chambers immediately or store in the incubator for future use (two weeks).</li>
	<li>Count the cell density of the differentiated HL60 cells with a hemocytometer. 
	NOTE: The cell density often reaches about 1.0 x 106 cells/ml. Cell density greater than 3.0 x 106 cells/ml usually gives undesirable transfection efficiency. As HL60 cells are suspension cells, no resuspension is required.</li>
	<li>Centrifuge the cells at 200 x g for 5 min at RT. Remove the supernatant and resuspend 2 x 106 HL60 cells in 100 &#956;l of transfection reagent.</li>
	<li>Add 4 &#956;g of GFP-PKD1 plasmid9 into the 100 &#956;l mixture of cells and transfection reagents and mix gently and thoroughly. Add the mixture into the cuvette provided.</li>
	<li>Select the manufacturer&#39;s pre-set program for the human leukocyte cell line and perform the electroporation. 
	NOTE: Further information is available from the manufacturer&#39;s website.</li>
	<li>After electroporation, immediately and gently add pre-warmed 500 &#956;l RPMI 6140 electroporation recovery medium to the cells, and gently transfer the cells from the cuvette to a 1.6ml tube with the plastic pipettes provided.</li>
	<li>Incubate the cells for 30 min in a humidified 37 &#176;C incubator with 5% CO2. Seed 100 &#956;l or 500 &#956;l of cells into one well of a 4-well or a 1-well chamber, respectively.</li>
	<li>Add RPMI 1640 culture medium to a final volume of 1 ml or 4 ml to that same well of a 4-well or a 1-well chamber, respectively. Incubate the cells in a humidified 37 &#176;C incubator with 5 % CO2 for 3 hr. Pre-warm RPMI 1640 starving medium to 37 &#176;C.</li>
	<li>Using a 1ml pipette, gently remove 0.8 ml or 3 ml of RPMI 1640 culture medium from the well of a 4-well chamber or a 1-well chamber, respectively. Be gentle and avoid sucking away adhering HL60 cells. 
	NOTE: HL60 cells grow and are differentiated in suspension media. After being differentiated or with certain treatments, HL60 cells adhere to the substratum coated with polylysine, collagen, gelatin, or fibronectin.</li>
	<li>Add 0.8 ml or 3 ml of RPMI 1640 starving medium into the well of a 4-well or 1-well chamber, respectively. Wait 1 hr. 
	NOTE: At this point the cells are ready for imaging experiments9.</li>
</ol>
5. Monitoring GPCR-mediated Membrane Translocation of PKD1 by Multi-channel Fluorescent Microscopy
<ol>
	<li>Prepare a fresh mixture of 100 nM fMLP and 1 &#956;g/ml Alexa 594 in RPMI 1640 starving medium as stimuli. 
	NOTE: The mixture of fMLP and Alexa 594 needs to be freshly made to ensure maximum efficacy.</li>
	<li>Mount one 4-well chamber seeded with cells over a 40X oil lens.</li>
	<li>Set the Fluorescent Microscope in the Multichannel Configuration as Follows:
	<ol>
		<li>Set the green emission channel (500-530 nm) for GFP-tagged PKD1, red emission channel (580-620 nm) for chemoattractant fMLP (Alexa 594), and transmitted light channel for identifying adhering HL60 cells.</li>
		<li>Start &#34;live&#34; acquisition and optimize the acquisition conditions of the three channels:</li>
		<li>Fine-tune between intensity and laser power for all three channels to minimize photobleach for a longer period of imaging.</li>
		<li>If necessary, average every 2 to 4 frames to decrease the background noise ratio for better quality of images. Use a 1 sec interval.</li>
	</ol>
	</li>
	<li>Search for adhering cells that express strong PKD1-GFP in the view of the GFP and DIC (differential interference contrast) channel. Adhering cells are morphologically flat and do not move rapidly.</li>
	<li>After identifying adhering HL60 cells that highly express GFP-PKD1, optimize the acquisition condition by lowering the laser power while increasing the detector gain for each channel to decrease the photobleach for long-term imaging (Figure 3A).</li>
	<li>Take up 100 &#956;l of fMLP/Alexa 594 solution with a 200 &#956;l pipetter and set aside. Select the &#34;time-lapse acquisition&#34; and set the intervals to 2 sec and the number of frames to acquire to 100. 
	NOTE: the intervals and number of frames to acquire depend on the purpose of experiments. 1-2 sec intervals and 100 frames are often used for imaging experiments, as in Figure 3. For slowly migrating cells, longer intervals are necessary for long-term imaging. In contrast, for rapidly migrating cells, shorter intervals are required for observing the continuous details of the cell migration.</li>
	<li>Carefully take off the lid of a 4-well chamber so that the position of the chamber is not changed. Start acquisition immediately and acquire 3-5 images of the unstimulated cells while positioning the 200 &#956;l pipetter directly over the cells using the spot of laser light as a guide.</li>
	<li>Pipette the fMLP/Alexa 594 mixture over the cells being imaged with a quick and even flow and finish the complete set of time-lapse acquisition. Name and save the data that will be subjected to further data analysis.</li>
</ol>
6. Imaging Chemotaxing Cells in Visible and Controllable Chemo-attractant Stimuli
<ol>
	<li>Using a 10 &#956;l micropipetter and injection tips, backfill a micropipette with 30 &#956;l freshly prepared mixture of fMLP and Alexa594.</li>
	<li>Attach the micropipette to a micropipette holder mounted on top of a microscope stage and connect the tubing to the pressure supply unit, an apparatus that provides a constant pressure to release fMLP/Alexa594 solution from the micropipette to generate a steady gradient.</li>
	<li>Turn on the pressure supply and set the compensation pressure (Pc) to 70 hPa.</li>
	<li>Mount a 1-well chambered coverslip seeded with HL60 cells expressing PKD1-GFP over a 40X oil lens on a confocal microscope or its equivalent fluorescent microscope.</li>
	<li>Set acquisition in channel mode in the following configuration: green channel (500-530 nm) for GFP-tagged PKD1; red channel (580-620 nm) for chemoattractant fMLP (Alexa594); and transmitted light channel for identifying adhering HL60 cells.</li>
	<li>Start &#34;live&#34; acquisition and optimize the acquisition conditions for the three channels: adjust the intensities of each channel for quantitative analysis of imaging data; fine-tune between intensity and laser power for all three channels to obtain images with the best quality and minimize the photo-bleach. If necessary, average every 2 to 4 frames for better image quality. We usually use a 1 sec interval.</li>
	<li>Start &#34;live&#34; acquisition and search for adhering cells that express strong PKD1-GFP in the view of the GFP and transmitted light channels. Using bright-field optics, center the micropipette in the center field of view (Figure 4A) in order to visualize the fMLP gradient and the adhering HL60 cells expressing PKD1-GFP.</li>
	<li>Select time-lapse acquisition and set the intervals to 2 sec intervals and the number of frames to acquire to 100. 
	NOTE: After collecting each time-lapse series, name and save the data that will be subjected to further data analysis.</li>
</ol>

<ol>
	<li>Pocha, S. M., Montell, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+and+molecular+mechanisms+of+single+and+collective+cell+migrations+in+Drosophila:+themes+and+variations.">Cellular and molecular mechanisms of single and collective cell migrations in Drosophila: themes and variations.</a> Annual review of genetics. 48, 295-318 (2014).</li><li>Wen, Z., Zheng, J. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directional+guidance+of+nerve+growth+cones.">Directional guidance of nerve growth cones.</a> Current opinion in neurobiology. 16, 52-58 (2006).</li><li>Bravo-Cordero, J. J., Hodgson, L., Condeelis, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directed+cell+invasion+and+migration+during+metastasis.">Directed cell invasion and migration during metastasis.</a> Current opinion in cell biology. 24, 277-283 (2012).</li><li>Zigmond, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemotaxis+by+polymorphonuclear+leukocytes.">Chemotaxis by polymorphonuclear leukocytes.</a> The Journal of cell biology. 77, 269-287 (1978).</li><li>Jin, T., Xu, X., Hereld, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemotaxis,+receptors+and+human+disease.">Chemotaxis, receptors and human disease.</a> Cytokine. 44, 1-8 (2008).</li><li>Dong, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=P-Rex1+is+a+primary+Rac2+guanine+nucleotide+exchange+factor+in+mouse+neutrophils.">P-Rex1 is a primary Rac2 guanine nucleotide exchange factor in mouse neutrophils.</a> Current biology : CB. 15, 1874-1879 (2005).</li><li>Li, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directional+sensing+requires+G+beta+gamma-mediated+PAK1+and+PIX+alpha-dependent+activation+of+Cdc42.">Directional sensing requires G beta gamma-mediated PAK1 and PIX alpha-dependent activation of Cdc42.</a> Cell. 114, 215-227 (2003).</li><li>Van Haastert, P. J., Devreotes, P. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemotaxis:+signalling+the+way+forward.">Chemotaxis: signalling the way forward.</a> Nature reviews: Molecular cell biology. 5, 626-634 (2004).</li><li>Xu, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GPCR-Mediated+PLCbetagamma/PKCbeta/PKD+Signaling+Pathway+Regulates+the+Cofilin+Phosphatase+Slingshot+2+in+Neutrophil+Chemotaxis.">GPCR-Mediated PLCbetagamma/PKCbeta/PKD Signaling Pathway Regulates the Cofilin Phosphatase Slingshot 2 in Neutrophil Chemotaxis.</a> Mol Biol Cell. , (2015).</li><li>Boyden, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+chemotactic+effect+of+mixtures+of+antibody+and+antigen+on+polymorphonuclear+leucocytes.">The chemotactic effect of mixtures of antibody and antigen on polymorphonuclear leucocytes.</a> The Journal of experimental medicine. 115, 453-466 (1962).</li><li>Zigmond, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ability+of+polymorphonuclear+leukocytes+to+orient+in+gradients+of+chemotactic+factors.">Ability of polymorphonuclear leukocytes to orient in gradients of chemotactic factors.</a> The Journal of cell biology. 75, 606-616 (1977).</li><li>Zicha, D., Dunn, G. A., Brown, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+direct-viewing+chemotaxis+chamber.">A new direct-viewing chemotaxis chamber.</a> Journal of cell science. 99 (Pt4), 769-775 (1991).</li><li>Muinonen-Martin, A. J., Veltman, D. M., Kalna, G., Insall, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+chamber+for+direct+visualisation+of+chemotaxis.">An improved chamber for direct visualisation of chemotaxis.</a> PloS one. 5, e15309 (2010).</li><li>Bozzaro, S., Fisher, P. R., Loomis, W., Satir, P., Segall, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guenther+Gerisch+and+Dictyostelium,+the+microbial+model+for+ameboid+motility+and+multicellular+morphogenesis.">Guenther Gerisch and Dictyostelium, the microbial model for ameboid motility and multicellular morphogenesis.</a> Trends in cell biology. 14, 585-588 (2004).</li><li>Xu, X., Jin, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+G-protein+coupled+receptor+(GPCR)-mediated+signaling+events+that+control+chemotaxis+of+Dictyostelium+discoideum.">Imaging G-protein coupled receptor (GPCR)-mediated signaling events that control chemotaxis of Dictyostelium discoideum.</a> Journal of visualized experiments : JoVE. , (2011).</li><li>Lin, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+dynamic+temporal+and+spatial+concentration+gradients+using+microfluidic+devices.">Generation of dynamic temporal and spatial concentration gradients using microfluidic devices.</a> Lab Chip. 4, 164-167 (2004).</li><li>Eiseler, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+kinase+D1+regulates+cofilin-mediated+F-actin+reorganization+and+cell+motility+through+slingshot.">Protein kinase D1 regulates cofilin-mediated F-actin reorganization and cell motility through slingshot.</a> Nature cell biology. 11, 545-556 (2009).</li><li>Roddie, P. H., Paterson, T., Turner, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+transfer+to+primary+acute+myeloid+leukaemia+blasts+and+myeloid+leukaemia+cell+lines.">Gene transfer to primary acute myeloid leukaemia blasts and myeloid leukaemia cell lines.</a> Cytokines Cell Mol Ther. 6, 127-134 (2000).</li><li>Schakowski, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+non-viral+method+for+transfection+of+primary+leukemia+cells+and+cell+lines.">Novel non-viral method for transfection of primary leukemia cells and cell lines.</a> Genet Vaccines Ther. 2, 1 (2004).</li><li>Uchida, E., Mizuguchi, H., Ishii-Watabe, A., Hayakawa, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+the+efficiency+and+safety+of+non-viral+vector-mediated+gene+transfer+into+a+wide+range+of+human+cells.">Comparison of the efficiency and safety of non-viral vector-mediated gene transfer into a wide range of human cells.</a> Biol Pharm Bull. 25, 891-897 (2002).</li></ol>

The authors declare no competing financial interest for this work.

In the present study, we show two examples of chemotaxis assays: first, simultaneous monitoring of multiple chemotaxis assays by the cell mobility analysis device; and second, visualization of the chemoattractant gradient and the spatiotemporal dynamics of signaling events in the same cells in real time.
Cell mobility analysis device for multiple simultaneous chemotaxis assays
In the present study, we introduced a detailed protocol to perform multiple simultaneous chemotaxis assays using a cell mobility analysis device. This device allows user to observe cellular chemotaxis behavior with a 10X objective lens in conventional bright-field observation. Because of the diffusion-dominant characteristics of the fluid flow, the cell mobility analysis device generates highly predictable, stable gradients and allows up to six chemotaxis assays to be carried out simultaneously. The critical steps of the protocol are to obtain reliable gradients and to align the cells on the terrace line. The user should strictly follow the manufacturer&#39;s instructions for holder assembly and injection of chemoattractants and cells. Detailed instructions are also available online. Compared to alternative chemotaxis methods11-13,15, this device significantly improves the reliability and efficiency of chemotaxis assays. Four sizes of cell mobility analysis device chip in sizes of 4, 5, 6, and 8 &#956;m are available to accommodate different types and sizes of cells. We found that a 4 or 5 &#956;m cell mobility analysis device chip is suitable for the HL60 and D. discoideum cells, which are about 10-15 &#956;m in diameter. However, one limitation is that this device is not suitable for all types of cells. We had little success using the cell mobility analysis device chemotaxis assay with Raw267.4 cells. The reason may be that Raw267.4 cells migrate too slowly. The time required for efficient chemotaxis of Raw267.4 cells might be much longer than the time a gradient is maintained by the device. Instead, a transwell migration assay worked well for Raw267.4 cells9. Another limitation is that fluorescent observation is not possible with the current device. A future direction is to monitor fluorescent imaging with higher magnification. This is possible with the improved cell mobility analysis device, which is equipped with fluorescent detection and a 100X objective lens. In addition, the number of simultaneous assays is also increased to 12. All these improvements facilitate the enhanced throughput of chemotaxis assays and the observation of subcellular dynamics in migrating cells.
High transfection efficiency of HL60 cells to express fluorescent protein-tagged protein
HL60 cells are an actively dividing leukemia cell line and grow in suspension. As previously reported18-20, HL60 cells are resistant to gene transfer. Both lipid and electroporation gene transfer have been tested, and higher transfection efficiency was obtained with electroporation, as described in detail in section 4. Obtaining high viability after electroporation is critical for obtaining high transfection efficiency because of the severe damage that results from electroporation. Subsequently, thorough and gentle cell handling are required, especially after electroporation. All media have to be pre-warmed and gently added to the cells in any steps after electroporation. It is also critical to minimize the exposure time of cells to the electroporation reagent. To avoid cell death, after electroporation, RPMI 1640 electroporation recovery medium must be added to the cells immediately. We found that 20% FBS in the recovery medium gives a much higher cell recovery rate than 10% FBS. After electroporation, incubation in RPMI 1640 electroporation recovery medium for 30 min is critical for greater viability and transfection efficiency. To further increase transfection efficiency, we also used 4 &#956;g plasmid DNA per transfection, which is almost the twice amount of plasmid recommended by the manufacturer.
There are two major limitations on electroporation transfection: the transiency of the protein expression and the cell number limit (2 x 106 cells per transfection). In undifferentiated cells, expression is detectable only during the first couple of cell divisions, since the vector plasmid is diluted by half after each cell division. For The differentiated HL60 cells survive no more than 48 hr. As a result, any experiment requires fresh transfections 6 hr prior to the experiment. The cell number for one electroporation merely meets the minimum requirement for any biochemical assays. For the purpose of repetitive use or large quantity, it is strongly recommended that an undifferentiated HL60 cell line be established that stably expresses the protein of interest which has been tagged with a fluorescent protein by a viral vector, if a viral vector is available or can be constructed.

Eukaryotic cells sense and move toward the higher concentration within a chemoattractant gradient, a cellular process referred as chemotaxis. Chemotaxis plays critical roles in many physiological processes, such as embryogenesis1, neuron patterning2, metastasis of cancer cells3, recruitment of neutrophils to sites of inflammation4, and the development of the model organism Dictyostelium discoideum5. In general, eukaryotic cells sense chemoattractants using G protein-coupled receptors5. The engagement of chemoattractants with these receptors promotes dissociation of the heterotrimeric G proteins G&#945; and G&#946;&#947;, which in turn activate downstream signal transduction pathways that ultimately regulate the spatiotemporal organization of the actin cytoskeleton to drive cell migration5-9.
Cell biologists have been developing and improving chemotaxis assays to examine how G protein-coupled receptor (GPCR) signaling mediates directed cell migration. The Boyden chamber or transwell migration assay was developed in 1960 by Boyden10. The assay works by creating a gradient of chemoattractant compounds between two wells that are separated by a microporous membrane. Its simplicity and ease of use make it the most widely used chemotaxis assay to date. However, the assay does not enable the migrating process of the cells to be visualized. The Zigmond chamber is the first visual microfluidic device that allows clear imaging of cell migration on a coverslip across a narrow constriction toward a source chemoattractant11. Dunn12 and Insall13 modified and improved the high-resolution and long-term imaging capability of the Zigmond chamber chemotaxis assay. Because of the highly predictable, diffusion-dominant characteristics of fluid flow, microfluidics has been providing solutions for next-generation chemotaxis assays such as EZ-TAXIScan (a cell mobility analysis device).
With the stability of the gradient ensured, the device allows six chemotaxis assays to be carried out simultaneously (Figure 1A). In contrast to the directionally fixed gradients generated in the various chamber assays above, the needle or micropipette assay developed by Guenther Gerisch generates a gradient with a movable source14. In the assay, chemoattractant is released from a movable micropipette to generate a stable gradient. With this needle assay, researchers found that different cells generate pseudopods with fundamentally different characteristics. Applying fluorescent microscopy, we were able to visualize the gradient to facilitate its quantitative measurement throughout15. In this study, we describe detailed methods for preparing chemotactic HL60 (human promyelocytic leukemia) cells, simultaneously monitoring multiple chemotaxis assays with the cell mobility analysis device, and visualizing the GPCR-mediated spatiotemporal dynamics of signaling molecules such as protein kinase D1 in single live cells in response to visible, spatiotemporally controllable chemoattractant stimuli. Our advanced imaging methods can be applied to general chemotaxis studies, and are especially suitable for mammalian cell systems.

<table border="0" cellpadding="0" cellspacing="0" width="459">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="61">
			<td height="61" style="height:61px;width:120px;">RPMI 1640 Medium GlutaMAX</td>
			<td style="width:107px;">Life technologies</td>
			<td style="width:120px;">61870-036</td>
			<td style="width:113px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:120px;">Sodium pyruvate</td>
			<td style="width:107px;">Thermo Fisher Scietific</td>
			<td style="width:120px;">11360-070</td>
			<td style="width:113px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:120px;">Fetal bovine serum</td>
			<td style="width:107px;">Gemini Bio-Products</td>
			<td style="width:120px;">100-106</td>
			<td style="width:113px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:120px;">1M HEPES sterile solution, pH7.3</td>
			<td style="width:107px;">Quality Biological Inc.</td>
			<td style="width:120px;">A611-J848-06</td>
			<td style="width:113px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:120px;">Penicillin streptomycin solution</td>
			<td style="width:107px;">Fisher Scientific</td>
			<td align="right" style="width:120px;">15140122</td>
			<td style="width:113px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:120px;">NucleofectorTM 2b</td>
			<td style="width:107px;">Lonza</td>
			<td style="width:120px;">AAB-1001</td>
			<td style="width:113px;"></td>
		</tr>
		<tr height="217">
			<td height="217" style="height:217px;width:120px;">AmaxaTM Cell Line NucleofectorTM Kit V including NucleofectorTM Solution, Singe use pipettes, AmaxaTM certified 100 ml aluminum electrode cuvettes</td>
			<td style="width:107px;">Lonza</td>
			<td style="width:120px;">VCA-1003</td>
			<td style="width:113px;"></td>
		</tr>
		<tr height="81">
			<td height="81" style="height:81px;width:120px;">Lab-Tek chambered #1.0 Borosilicate Coverglass</td>
			<td style="width:107px;">Nalge Nunc International Inc</td>
			<td align="right" style="width:120px;">155383</td>
			<td style="width:113px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:120px;">2 % Gelatin solution</td>
			<td style="width:107px;">Sigma-Aldrich</td>
			<td style="width:120px;">G1393</td>
			<td style="width:113px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:120px;">Fibronectin</td>
			<td style="width:107px;">Sigma-Aldrich</td>
			<td style="width:120px;">F1141</td>
			<td style="width:113px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:120px;">HBSS (Hanks&#8217; Balanced Salt Solution)</td>
			<td style="width:107px;">Life technologies</td>
			<td style="width:120px;">14025-076</td>
			<td style="width:113px;"></td>
		</tr>
		<tr height="39">
			<td height="60" rowspan="2" style="height:60px;width:120px;">Bovine serum albumin</td>
			<td rowspan="2" style="width:107px;">Sigma-Aldrich</td>
			<td rowspan="2" style="width:120px;">A3803</td>
			<td rowspan="2" style="width:113px;"></td>
		</tr>
		<tr height="21">
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:120px;">Single well Lab-Tek II coverglass chambers</td>
			<td style="width:107px;">Nalge Nunc International Inc</td>
			<td align="right" style="width:120px;">155361</td>
			<td style="width:113px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:120px;">Four-well Lab-Tek II coverglass chambers</td>
			<td style="width:107px;">Nalge Nunc International Inc</td>
			<td align="right" style="width:120px;">155383</td>
			<td style="width:113px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:120px;">Alexa 594</td>
			<td style="width:107px;">Thermo Fisher Scientific</td>
			<td style="width:120px;">A-10438</td>
			<td style="width:113px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:120px;">fMLP</td>
			<td style="width:107px;">Sigma -Aldrich</td>
			<td style="width:120px;">F3506-5MG</td>
			<td style="width:113px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:120px;">Cover glass thickness 2 22 x 22 mm</td>
			<td style="width:107px;">Corning</td>
			<td style="width:120px;">2855-22</td>
			<td style="width:113px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:120px;">EZ-TAXIScan</td>
			<td style="width:107px;">Effector Cell Institute, Inc.</td>
			<td style="width:120px;">MIC-1001</td>
			<td style="width:113px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:120px;">EZ-TAXIScan chip (5 mm)</td>
			<td style="width:107px;">Effector Cell Institute, Inc.</td>
			<td style="width:120px;">EZT-F01-5</td>
			<td style="width:113px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:120px;">1701RN 10ul syringe</td>
			<td style="width:107px;">Hamilton</td>
			<td align="right" style="width:120px;">80030</td>
			<td style="width:113px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:120px;">Femtotips II Injection tips</td>
			<td style="width:107px;">Eppendorf</td>
			<td align="right" style="width:120px;">5242956003</td>
			<td style="width:113px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:120px;">Femtotips II</td>
			<td style="width:107px;">Eppendorf</td>
			<td align="right" style="width:120px;">930000043</td>
			<td style="width:113px;"></td>
		</tr>
		<tr height="121">
			<td height="121" style="height:121px;width:120px;">TransferMan NK2, including motor module, X head with angle adjuster, and Positioning aids.&#160;</td>
			<td style="width:107px;">Eppendorf</td>
			<td align="right" style="width:120px;">5188900056</td>
			<td style="width:113px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:120px;">DIAS software</td>
			<td style="width:107px;">Solltech Inc.</td>
			<td style="width:120px;"></td>
			<td style="width:113px;"></td>
		</tr>
		<tr height="181">
			<td height="181" style="height:181px;width:120px;">LSM 780 META or equivalent confocal microscope with a 40X 1.3 NA or 60X 1.4 NA oil DIC Plan-Neofluar objective lens</td>
			<td style="width:107px;">Carl Zeiss</td>
			<td style="width:120px;"></td>
			<td style="width:113px;"></td>
		</tr>
	</tbody>
</table>

imaging g protein-coupled receptor-mediated chemotaxis and its signaling events in neutrophil-like hl60 cells

Eukaryotic cells sense and move towards a chemoattractant gradient, a cellular process referred as chemotaxis. Chemotaxis plays critical roles in many physiological processes, such as embryogenesis, neuron patterning, metastasis of cancer cells, recruitment of neutrophils to sites of inflammation, and the development of the model organism Dictyostelium discoideum. Eukaryotic cells sense chemo-attractants using G protein-coupled receptors. Visual chemotaxis assays are essential for a better understanding of how eukaryotic cells control chemoattractant-mediated directional cell migration. Here, we describe detailed methods for: 1) real-time, high-resolution monitoring of multiple chemotaxis assays, and 2) simultaneously visualizing the chemoattractant gradient and the spatiotemporal dynamics of signaling events in neutrophil-like HL60 cells.

Simultaneous imaging of chemotaxis of multiple HL60 cells using cell mobility analysis device
Based on the principle of microfluidics16, the manufacturer has provided simulated profiles of gradients: a gradient is generated within 1 min, stabilized within 5 min, and maintained over 2 hr. The highly predictable profiles of the stable gradients generated by microfluidics allow multiple chemotaxis assays to be carried out simultaneously. In the present study, we observed three simultaneous chemotaxis assays (Figure 2A and Movie 1). We found that HL60 cells started chemotaxing immediately after the chemoattractant was injected into the well of the chemoattractant, and kept chemotaxing in a straight path for the following 60 min, consistent with the simulation results for gradient stability. Tracing the travel path and morphology of the cells allows quantitative measurements and subsequent comparison of the chemotaxis behaviors using a chemotaxis index that includes total path length, speed, directionality, and roundness of the cells (Figure 2B). Total path length is the sum of the lengths of the line segments connecting the centroids of the path. Speed is obtained by dividing the total path length by the time. Directionality is measured upward and is defined as: (Y coordinate of the end of the path minus Y coordinate of the beginning) divided by total path length. This gives 1.0 for an object moving directly upward. The roundness of the cell is a measure (in percent) of how efficiently a given amount of perimeter encloses area. A circle has the largest area for any given perimeter and has a roundness parameter of 100%. A straight line encloses no area and has a roundness parameter of 0%. We show the quantitatively measured chemotaxis behavior as described by the selected chemotaxis parameters (Figure 2C).
Monitoring PKD subcellular localization in HL60 cells under a spatiotemporally visible and controllable fMLP stimulus
It is a great technical advance to apply fluorescently labeled and controllable chemoattractant stimulation to an experimental system. Historically, we have applied either homogeneous (also called uniform) stimulation or gradient stimulation to observe cell response and behaviors. However, &#34;blind&#34; stimulation not only provides no spatiotemporal information on how the stimulus reaches the cells, but also casts doubt on any &#34;abnormal&#34; observations of cell response to stimulation, simply because we do not see the stimulus. We have previously shown that fluorescent dye (Alexa594) can be applied with chemoattractant to establish a linear relationship between chemoattractant concentration and monitored fluorescent dye intensity15. With an acquisition configuration of green fluorescent protein (GFP), a red emission of fluorescent dye (Alexa594), and transmitted light, we are able to monitor the adhering cells, the application of the stimulus, and the cell response to the stimulus (Figure 3A). Protein kinase D is a family of serine/threonine kinases that play essential roles in directed cell migration9,17. In response to uniformly applied fMLP (red) stimulation, HL60 cells mediate a robust membrane translocation of GFP-tagged protein kinase D1 (green) (Figure 3B and Movie 2). In an fMLP gradient (red) (Figure 4A), HL60 cells actively recruit PKD1 to the leading edge (Figure 4B and Movie 3). A close comparison of the subcellular localization of GFP in the protrusion of the leading edge indicates that PKD1 localizes at the rear of the leading edge (Figure 4C).
<img alt="Figure 1" src="/files/ftp_upload/54511/54511fig1.jpg" /> 
Figure 1. Cell mobility analysis device allows up to 6 simultaneous chemotaxis assays. (A) Scheme shows the design of a cell mobility analysis device chip for simultaneous monitoring of 6 independent chemotaxis assays. Red shows chemoattractant added to the wells. (B) Introduction of HL60 cells to the wells of cells while the chemoattractant diffuses to establish a steady fMLP gradient. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54511/54511fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54511/54511fig2.jpg" /> 
Figure 2. Simultaneous monitoring of multiple chemotaxis assays with HL60 cells. (A) Montage shows images of cell mobility analysis device chemotaxis assay to examine the inhibitory effects of PKD-specific inhibitors on chemotaxis at times of 0 and 12 min after gradient application. Chemotactic HL60 cells were pre-treated with PKD inhibitor 1 &#956;M CID755673 for 30 min. HL60 cells with or without the treatment of PKD inhibitor were allowed to chemotax in either RPMI1640 starving medium or 100 nM fMLP gradients for 12 min. (B) Scheme shows the travel path length and morphology of traced HL60 cells. (C) Quantification of chemotaxis as total path length, speed, directionality, and roundness. Mean &#177; SD is shown; n = 10, 12, or 11 for no gradient, fMLP gradient without CID755673 treatment, and fMLP treatment with CID755673 treatment, respectively. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54511/54511fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54511/54511fig3.jpg" /> 
Figure 3. GPCR-mediated robust membrane translocation of PKD1 in response to uniformly applied fMLP stimuli. (A) Multichannel monitoring of PKD1-GFP (green), chemoattractant (1 &#956;M fMLP mixed with 0.1 &#956;g/ml fluorescent dye Alexa 594, red), and DIC (differential interference contrast) to identify the adhering HL60 cells in a well of a 4well chamber coated with 0.2% gelatin in RPMI 1640 medium. Scale bar = 10 &#956;m. (B) Montage shows that uniformly applied fMLP (red) induces robust membrane translocation of PKD1-GFP (green). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54511/54511fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/54511/54511fig4.jpg" /> 
Figure 4. Leading edge localization of PKD1 in chemotaxing HL60 cells. (A) Channel mode acquisition configuration facilitates the visualization of the fMLP gradient and the spatiotemporal dynamics of PKD1. In A-C, HL60 cells transiently expressed GFP-tagged PKD1; to visualize the fMLP gradient generated from a micropipette (DIC), 100 nM fMLP (Red) was mixed with 0.1 &#956;g/ml fluorescent dye Alexa 594. (B) Enriched localization of PKD1 at the leading edge of the chemotaxing cell. Scale bar = 10 &#956;m. (C) Merged images show that PKD1 localizes at the rear of the leading edge in HL60 cells. Green shows PKD1 cellular localization, and the DIC image shows the protruding area of the leading edge. Scale bar = 5 &#956;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54511/54511fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Imaging G Protein-coupled Receptor-mediated Chemotaxis and its Signaling Events in Neutrophil-like HL60 Cells at JoVE.com

Imaging G Protein-coupled Receptor-mediated Chemotaxis and its Signaling Events in Neutrophil-like HL60 Cells

Visual chemotaxis assays are essential for a better understanding of how eukaryotic cells control chemoattractant-mediated directional cell migration. Here, we describe detailed methods for: 1) real-time, high-resolution monitoring of multiple chemotaxis assays, and 2) simultaneously visualizing the chemoattractant gradient and the spatiotemporal dynamics of signaling events in neutrophil-like HL60 cells.

Eukaryotic cells sense and move towards a chemoattractant gradient, a cellular process referred as chemotaxis. Chemotaxis plays critical roles in many ...

imaging-g-protein-coupled-receptor-mediated-chemotaxis-its-signaling

Nanyang Technological University

Research

JoVE Journal

Biology

10.0K Views.  National Institute of Allergy and Infectious Diseases, National Institutes of Health. Visual chemotaxis assays are essential for a better understanding of how eukaryotic cells control chemoattractant-mediated directional cell migration. Here, we describe detailed methods for: 1) real-time, high-resolution monitoring of multiple chemotaxis assays, and 2) simultaneously visualizing the chemoattractant gradient and the spatiotemporal dynamics of signaling events in neutrophil-like HL60 cells.

Video: Imaging G Protein-coupled Receptor-mediated Chemotaxis and its Signaling Events in Neutrophil-like HL60 Cells

Implantation of Ferumoxides Labeled Human Mesenchymal Stem Cells in Cartilage Defects

The field of tissue engineering integrates the principles of engineering, cell biology and medicine towards the regeneration of specific cells and functional tissue. Matrix associated stem cell implants (MASI) aim to regenerate cartilage defects due to arthritic or traumatic joint injuries. Adult mesenchymal stem cells (MSCs) have the ability to differentiate into cells of the chondrogenic lineage and have shown promising results for cell-based articular cartilage repair technologies. Autologous MSCs can be isolated from a variety of tissues, can be expanded in cell cultures without losing their differentiation potential, and have demonstrated chondrogenic differentiation in vitro and in vivo1, 2.
In order to provide local retention and viability of transplanted MSCs in cartilage defects, a scaffold is needed, which also supports subsequent differentiation and proliferation. The architecture of the scaffold guides tissue formation and permits the extracellular matrix, produced by the stem cells, to expand. Previous investigations have shown that a 2% agarose scaffold may support the development of stable hyaline cartilage and does not induce immune responses3.
Long term retention of transplanted stem cells in MASI is critical for cartilage regeneration. Labeling of MSCs with iron oxide nanoparticles allows for long-term in vivo tracking with non-invasive MR imaging techniques4.
This presentation will demonstrate techniques for labeling MSCs with iron oxide nanoparticles, the generation of cell-agarose constructs and implantation of these constructs into cartilage defects. The labeled constructs can be tracked non-invasively with MR-Imaging.

Goal of the presentation is to demonstrate a highly reproducible method to generate matrix associated stem cell implants in cartilage defects, which can be visualized with MR imaging. Stem cells are labeled with FDA-approved Ferumoxides, mixed with agarose, implanted into cartilage defects and imaged with a 7T MR scanner.

The field of tissue engineering integrates the principles of engineering, cell biology and medicine towards the regeneration of specific cells and ...

In vivo Quantification of G Protein Coupled Receptor Interactions using Spectrally Resolved Two-photon Microscopy

The study of protein interactions in living cells is an important area of research because the information accumulated both benefits industrial applications as well as increases basic fundamental biological knowledge. F&ouml;rster (Fluorescence) Resonance Energy Transfer (FRET) between a donor molecule in an electronically excited state and a nearby acceptor molecule has been frequently utilized for studies of protein-protein interactions in living cells. The proteins of interest are tagged with two different types of fluorescent probes and expressed in biological cells. The fluorescent probes are then excited, typically using laser light, and the spectral properties of the fluorescence emission emanating from the fluorescent probes is collected and analyzed. Information regarding the degree of the protein interactions is embedded in the spectral emission data. Typically, the cell must be scanned a number of times in order to accumulate enough spectral information to accurately quantify the extent of the protein interactions for each region of interest within the cell. However, the molecular composition of these regions may change during the course of the acquisition process, limiting the spatial determination of the quantitative values of the apparent FRET efficiencies to an average over entire cells. By means of a spectrally resolved two-photon microscope, we are able to obtain a full set of spectrally resolved images after only one complete excitation scan of the sample of interest. From this pixel-level spectral data, a map of FRET efficiencies throughout the cell is calculated. By applying a simple theory of FRET in oligomeric complexes to the experimentally obtained distribution of FRET efficiencies throughout the cell, a single spectrally resolved scan reveals stoichiometric and structural information about the oligomer complex under study. Here we describe the procedure of preparing biological cells (the yeast Saccharomyces cerevisiae) expressing membrane receptors (sterile 2 &alpha;-factor receptors) tagged with two different types of fluorescent probes. Furthermore, we illustrate critical factors involved in collecting fluorescence data using the spectrally resolved two-photon microscopy imaging system. The use of this protocol may be extended to study any type of protein which can be expressed in a living cell with a fluorescent marker attached to it.

In vivo Quantification of G Protein Coupled Receptor Interactions using Spectrally Resolved Two-photon Microscopy

By employing a spectrally resolved two-photon microscopy imaging system, pixel-level maps of F&ouml;rster Resonance Energy Transfer (FRET) efficiencies are obtained for cells expressing membrane receptors hypothesized to form homo-oligomeric complexes. From the FRET efficiency maps, we are able to estimate stoichiometric information about the oligomer complex under study.

The study of protein interactions in living cells is an important area of research because the information accumulated both benefits industrial ...

Imaging G-protein Coupled Receptor (GPCR)-mediated Signaling Events that Control Chemotaxis of Dictyostelium Discoideum

Many eukaryotic cells can detect gradients of chemical signals in their environments and migrate accordingly 1. This guided cell migration is referred as chemotaxis, which is essential for various cells to carry out their functions such as trafficking of immune cells and patterning of neuronal cells 2, 3. A large family of G-protein coupled receptors (GPCRs) detects variable small peptides, known as chemokines, to direct cell migration in vivo 4. The final goal of chemotaxis research is to understand how a GPCR machinery senses chemokine gradients and controls signaling events leading to chemotaxis. To this end, we use imaging techniques to monitor, in real time, spatiotemporal concentrations of chemoattractants, cell movement in a gradient of chemoattractant, GPCR mediated activation of heterotrimeric G-protein, and intracellular signaling events involved in chemotaxis of eukaryotic cells 5-8. The simple eukaryotic organism, Dictyostelium discoideum, displays chemotaxic behaviors that are similar to those of leukocytes, and D. discoideum is a key model system for studying eukaryotic chemotaxis. As free-living amoebae, D. discoideum cells divide in rich medium. Upon starvation, cells enter a developmental program in which they aggregate through cAMP-mediated chemotaxis to form multicullular structures. Many components involved in chemotaxis to cAMP have been identified in D. discoideum. The binding of cAMP to a GPCR (cAR1) induces dissociation of heterotrimeric G-proteins into G&gamma; and G&beta;&gamma; subunits 7, 9, 10. G&beta;&gamma; subunits activate Ras, which in turn activates PI3K, converting PIP2 into PIP3 on the cell membrane 11-13. PIP3 serve as binding sites for proteins with pleckstrin Homology (PH) domains, thus recruiting these proteins to the membrane 14, 15. Activation of cAR1 receptors also controls the membrane associations of PTEN, which dephosphorylates PIP3 to PIP2 16, 17. The molecular mechanisms are evolutionarily conserved in chemokine GPCR-mediated chemotaxis of human cells such as neutrophils 18. We present following methods for studying chemotaxis of D. discoideum cells. 1. Preparation of chemotactic component cells. 2. Imaging chemotaxis of cells in a cAMP gradient. 3. Monitoring a GPCR induced activation of heterotrimeric G-protein in single live cells. 4. Imaging chemoattractant-triggered dynamic PIP3 responses in single live cells in real time. Our developed imaging methods can be applied to study chemotaxis of human leukocytes.

Imaging G-protein Coupled Receptor GPCR-mediated Signaling Events that Control Chemotaxis of Dictyostelium Discoideum

Here, we describe detailed live cell imaging methods for investigating chemotaxis. We present fluorescence microscopic methods to monitor spatiotemporal dynamics of signaling events in migrating cells. Measurement of signaling events permits us to further understand how a GPCR-signaling network achieves gradient sensing of chemoattractants and controls directional migration of eukaryotic cells.

Many eukaryotic cells can detect gradients of chemical signals in their environments and migrate accordingly 1.  This guided cell migration is referred as ...

Quantifying Agonist Activity at G Protein-coupled Receptors

When an agonist activates a population of G protein-coupled receptors (GPCRs), it elicits a signaling pathway that culminates in the response of the cell or tissue. This process can be analyzed at the level of a single receptor, a population of receptors, or a downstream response. Here we describe how to analyze the downstream response to obtain an estimate of the agonist affinity constant for the active state of single receptors.
Receptors behave as quantal switches that alternate between active and inactive states (Figure 1). The active state interacts with specific G proteins or other signaling partners. In the absence of ligands, the inactive state predominates. The binding of agonist increases the probability that the receptor will switch into the active state because its affinity constant for the active state (Kb) is much greater than that for the inactive state (Ka). The summation of the random outputs of all of the receptors in the population yields a constant level of receptor activation in time. The reciprocal of the concentration of agonist eliciting half-maximal receptor activation is equivalent to the observed affinity constant (Kobs), and the fraction of agonist-receptor complexes in the active state is defined as efficacy (&#949;) (Figure 2).
Methods for analyzing the downstream responses of GPCRs have been developed that enable the estimation of the Kobs and relative efficacy of an agonist 1,2. In this report, we show how to modify this analysis to estimate the agonist Kb value relative to that of another agonist. For assays that exhibit constitutive activity, we show how to estimate Kb in absolute units of M-1.
Our method of analyzing agonist concentration-response curves 3,4 consists of global nonlinear regression using the operational model 5. We describe a procedure using the software application, Prism (GraphPad Software, Inc., San Diego, CA). The analysis yields an estimate of the product of Kobs and a parameter proportional to efficacy (&tau;). The estimate of &tau;Kobs of one agonist, divided by that of another, is a relative measure of Kb (RAi) 6. For any receptor exhibiting constitutive activity, it is possible to estimate a parameter proportional to the efficacy of the free receptor complex (&tau;sys). In this case, the Kb value of an agonist is equivalent to &tau;Kobs/&tau;sys 3.
Our method is useful for determining the selectivity of an agonist for receptor subtypes and for quantifying agonist-receptor signaling through different G proteins.

A method for estimating the affinity constant of an agonist for the active state (Kb) of a G protein-coupled receptor is described. The analysis provides absolute or relative measures of Kb depending on whether constitutive receptor activation is measurable. Our method applies to various responses downstream from receptor activation.

When an agonist activates a population of G protein-coupled receptors (GPCRs), it elicits a signaling pathway that culminates in the response of the cell ...

FRET Microscopy for Real-time Monitoring of Signaling Events in Live Cells Using Unimolecular Biosensors

F&ouml;rster resonance energy transfer (FRET) microscopy continues to gain increasing interest as a technique for real-time monitoring of biochemical and signaling events in live cells and tissues. Compared to classical biochemical methods, this novel technology is characterized by high temporal and spatial resolution. FRET experiments use various genetically-encoded biosensors which can be expressed and imaged over time in situ or in vivo1-2. Typical biosensors can either report protein-protein interactions by measuring FRET between a fluorophore-tagged pair of proteins or conformational changes in a single protein which harbors donor and acceptor fluorophores interconnected with a binding moiety for a molecule of interest3-4. Bimolecular biosensors for protein-protein interactions include, for example, constructs designed to monitor G-protein activation in cells5, while the unimolecular sensors measuring conformational changes are widely used to image second messengers such as calcium6, cAMP7-8, inositol phosphates9 and cGMP10-11. Here we describe how to build a customized epifluorescence FRET imaging system from single commercially available components and how to control the whole setup using the Micro-Manager freeware. This simple but powerful instrument is designed for routine or more sophisticated FRET measurements in live cells. Acquired images are processed using self-written plug-ins to visualize changes in FRET ratio in real-time during any experiments before being stored in a graphics format compatible with the build-in ImageJ freeware used for subsequent data analysis. This low-cost system is characterized by high flexibility and can be successfully used to monitor various biochemical events and signaling molecules by a plethora of available FRET biosensors in live cells and tissues. As an example, we demonstrate how to use this imaging system to perform real-time monitoring of cAMP in live 293A cells upon stimulation with a &beta;-adrenergic receptor agonist and blocker.

F&#246;rster resonance energy transfer (FRET) microscopy is a powerful technique for real-time monitoring of signaling events in live cells using various biosensors as reporters. Here we describe how to build a customized epifluorescence FRET imaging system from commercially available components and how to use it for FRET experiments.

F&ouml;rster resonance energy transfer (FRET) microscopy  continues to gain increasing interest as a technique for real-time monitoring  of biochemical ...

Live Cell Imaging of Early Autophagy Events: Omegasomes and Beyond

Autophagy is a cellular response triggered by the lack of nutrients, especially the absence of amino acids. Autophagy is defined by the formation of double membrane structures, called autophagosomes, that sequester cytoplasm, long-lived proteins and protein aggregates, defective organelles, and even viruses or bacteria. Autophagosomes eventually fuse with lysosomes leading to bulk degradation of their content, with the produced nutrients being recycled back to the cytoplasm. Therefore, autophagy is crucial for cell homeostasis, and dysregulation of autophagy can lead to disease, most notably neurodegeneration, ageing and cancer.	
	Autophagosome formation is a very elaborate process, for which cells have allocated a specific group of proteins, called the core autophagy machinery. The core autophagy machinery is functionally complemented by additional proteins involved in diverse cellular processes, e.g. in membrane trafficking, in mitochondrial and lysosomal biology. Coordination of these proteins for the formation and degradation of autophagosomes constitutes the highly dynamic and sophisticated response of autophagy. Live cell imaging allows one to follow the molecular contribution of each autophagy-related protein down to the level of a single autophagosome formation event and in real time, therefore this technique offers a high temporal and spatial resolution.	
	Here we use a cell line stably expressing GFP-DFCP1, to establish a spatial and temporal context for our analysis. DFCP1 marks omegasomes, which are precursor structures leading to autophagosomes formation. A protein of interest (POI) can be marked with either a red or cyan fluorescent tag. Different organelles, like the ER, mitochondria and lysosomes, are all involved in different steps of autophagosome formation, and can be marked using a specific tracker dye. Time-lapse microscopy of autophagy in this experimental set up, allows information to be extracted about the fourth dimension, i.e. time. Hence we can follow the contribution of the POI to autophagy in space and time.

Time-lapse microscopy of fluorescently labeled autophagy markers allows monitoring of the dynamic autophagy response with high temporal resolution. Using specific autophagy and organelle markers in a combination of 3 different colors, we can follow the contribution of a protein to autophagosome formation in a robust spatial and temporal context.

Autophagy is a  cellular response triggered by the lack of nutrients, especially the absence of  amino acids. Autophagy is defined by the formation of ...

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling processes. Extracting sterol-enriched membrane microdomains from plant cells for proteomic analysis is a difficult task mainly due to multiple preparation steps and sources for contaminations from other cellular compartments. The plasma membrane constitutes only about 5-20% of all the membranes in a plant cell, and therefore isolation of highly purified plasma membrane fraction is challenging. A frequently used method involves aqueous two-phase partitioning in polyethylene glycol and dextran, which yields plasma membrane vesicles with a purity of 95% 1. Sterol-rich membrane microdomains within the plasma membrane are insoluble upon treatment with cold nonionic detergents at alkaline pH. This detergent-resistant membrane fraction can be separated from the bulk plasma membrane by ultracentrifugation in a sucrose gradient 2. Subsequently, proteins can be extracted from the low density band of the sucrose gradient by methanol/chloroform precipitation. Extracted protein will then be trypsin digested, desalted and finally analyzed by LC-MS/MS. Our extraction protocol for sterol-rich microdomains is optimized for the preparation of clean detergent-resistant membrane fractions from Arabidopsis thaliana cell cultures.
We use full metabolic labeling of Arabidopsis thaliana suspension cell cultures with K15NO3 as the only nitrogen source for quantitative comparative proteomic studies following biological treatment of interest 3. By mixing equal ratios of labeled and unlabeled cell cultures for joint protein extraction the influence of preparation steps on final quantitative result is kept at a minimum. Also loss of material during extraction will affect both control and treatment samples in the same way, and therefore the ratio of light and heave peptide will remain constant. In the proposed method either labeled or unlabeled cell culture undergoes a biological treatment, while the other serves as control 4.

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Here we describe a robust method for the fractionation of plant plasma membranes into detergent resistant and detergent soluble membranes based on a mixture of unlabeled and in vivo fully 15N labeled Arabidopsis thaliana cell cultures. The procedure is applied for comparative proteomic studies to understand signaling processes.

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling ...

Genetically-encoded Molecular Probes to Study G Protein-coupled Receptors

To facilitate structural and dynamic studies of G protein-coupled receptor (GPCR) signaling complexes, new approaches are required to introduce informative probes or labels into expressed receptors that do not perturb receptor function. We used amber codon suppression technology to genetically-encode the unnatural amino acid, p-azido-L-phenylalanine (azF) at various targeted positions in GPCRs heterologously expressed in mammalian cells. The versatility of the azido group is illustrated here in different applications to study GPCRs in their native cellular environment or under detergent solubilized conditions. First, we demonstrate a cell-based targeted photocrosslinking technology to identify the residues in the ligand-binding pocket of GPCR where a tritium-labeled small-molecule ligand is crosslinked to a genetically-encoded azido amino acid. We then demonstrate site-specific modification of GPCRs by the bioorthogonal Staudinger-Bertozzi ligation reaction that targets the azido group using phosphine derivatives. We discuss a general strategy for targeted peptide-epitope tagging of expressed membrane proteins in-culture and its detection using a whole-cell-based ELISA approach. Finally, we show that azF-GPCRs can be selectively tagged with fluorescent probes. The methodologies discussed are general, in that they can in principle be applied to any amino acid position in any expressed GPCR to interrogate active signaling complexes.

We genetically-encode the unnatural amino acid, p-azido-L-phenylalanine at various targeted positions in GPCRs and show the versatility of the azido group in different applications. These include a targeted photocrosslinking technology to identify residues in the ligand-binding pocket of a GPCR, and site-specific bioorthogonal modification of GPCRs with a peptide-epitope tag or fluorescent probe.

To facilitate structural and dynamic studies of G protein-coupled receptor (GPCR) signaling complexes, new approaches are required to introduce ...

Identification of a Murine Erythroblast Subpopulation  Enriched in Enucleating Events by Multi-spectral Imaging Flow Cytometry

Erythropoiesis in mammals concludes with the dramatic process of enucleation that results in reticulocyte formation. The mechanism of enucleation has not yet been fully elucidated. A common problem encountered when studying the localization of key proteins and structures within enucleating erythroblasts by microscopy is the difficulty to observe a sufficient number of cells undergoing enucleation.&#160;We have developed a novel analysis protocol using multiparameter high-speed cell imaging in flow (Multi-Spectral Imaging Flow Cytometry), a method that combines immunofluorescent microscopy with flow cytometry, in order to identify efficiently a significant number of enucleating events, that allows to obtain measurements and perform statistical analysis.
We first describe here two in vitro erythropoiesis culture methods used in order to synchronize murine erythroblasts and increase the probability of capturing enucleation at the time of evaluation.&#160;Then, we describe in detail the staining of erythroblasts after fixation and permeabilization in order to study the localization of intracellular proteins or lipid rafts during enucleation by multi-spectral imaging flow cytometry.&#160;Along with size and DNA/Ter119 staining which are used to identify the orthochromatic erythroblasts, we utilize the parameters &#8220;aspect ratio&#8221; of a cell in the bright-field channel that aids in the recognition of elongated cells and &#8220;delta centroid XY Ter119/Draq5&#8221; that allows the identification of cellular events in which the center of Ter119 staining (nascent reticulocyte) is far apart from the center of Draq5 staining (nucleus undergoing extrusion), thus indicating a cell about to enucleate.&#160;The subset of the orthochromatic erythroblast population with high delta centroid and low aspect ratio is highly enriched in enucleating cells.

The present protocol describes a novel method of identifying a population of enucleating orthochromatic erythroblasts by multi-spectral imaging flow cytometry, providing a visualization of the erythroblast enucleation process.

Erythropoiesis in mammals concludes with the dramatic process of enucleation that results in reticulocyte formation. The mechanism of enucleation has not ...

Characterization of G Protein-coupled Receptors by a Fluorescence-based Calcium Mobilization Assay

For more than 20 years, reverse pharmacology has been the preeminent strategy to discover the activating ligands of orphan G protein-coupled receptors (GPCRs). The onset of a reverse pharmacology assay is the cloning and subsequent transfection of a GPCR of interest in a cellular expression system. The heterologous expressed receptor is then challenged with a compound library of candidate ligands to identify the receptor-activating ligand(s). Receptor activation can be assessed by measuring changes in concentration of second messenger reporter molecules, like calcium or cAMP. The fluorescence-based calcium mobilization assay described here is a frequently used medium-throughput reverse pharmacology assay. The orphan GPCR is transiently expressed in human embryonic kidney 293T (HEK293T) cells and a promiscuous G&#945;16 construct is co-transfected. Following ligand binding, activation of the G&#945;16 subunit induces the release of calcium from the endoplasmic reticulum. Prior to ligand screening, the receptor-expressing cells are loaded with a fluorescent calcium indicator, Fluo-4 acetoxymethyl. The fluorescent signal of Fluo-4 is negligible in cells under resting conditions, but can be amplified more than a 100-fold upon the interaction with calcium ions that are released after receptor activation. The described technique does not require the time-consuming establishment of stably transfected cell lines in which the transfected genetic material is integrated into the host cell genome. Instead, a transient transfection, generating temporary expression of the target gene, is sufficient to perform the screening assay. The setup allows medium-throughput screening of hundreds of compounds. Co-transfection of the promiscuous G&#945;16, which couples to most GPCRs, allows the intracellular signaling pathway to be redirected towards the release of calcium, regardless of the native signaling pathway in endogenous settings. The HEK293T cells are easy to handle and have proven their efficacy throughout the years in receptor deorphanization assays. However, optimization of the assay for specific receptors may remain necessary.

The here described fluorescence-based calcium mobilization assay is a medium-throughput reverse pharmacology screening system for the identification of functionally activating ligand(s) of orphan G protein-coupled receptors (GPCRs).

For more than 20 years, reverse pharmacology has been the preeminent strategy to discover the activating ligands of orphan G protein-coupled receptors ...