Name: fluorescence micropipette aspiration assay to investigate red blood cell mechanosensing
Uploaded: 2024-01-12T14:40:00.000+00:00
Duration: 7 min 2 s
Description: Watch this Scientific Journal Video about Advancing Live-Cell Mechanobiology Through Fluorescence Microaspiration at JoVE.com

We thank Nurul Aisha Zainal Abidin and Laura Moldovan for additional donor recruitment, blood collection, and phlebotomy support. We thank Tomas Anderson and Arian Nasser for organizing the equipment and reagents. This research was funded by the Australian Research Council (ARC) Discovery Project (DP200101970-L.A.J.); the National Health and Medical Research Council (NHMRC) of Australia Ideas Grant (APP2003904-L.A.J.); NHMRC Equipment Grant-L.A.J.; NSW Cardiovascular Capacity Building Program (Early-Mid Career Researcher Grant-L.A.J.); NSW CVRN-VCCRI Research Innovation Grant; Office of Global and Research Engagement (Sydney-Glasgow Partnership Collaboration Award-L.A.J.); L.A.J. is a National Heart Foundation Future Leader Fellow Level 2 (105863), and a Snow Medical Research Foundation Fellow (2022SF176).

This protocol follows the guidelines of and has been approved by the Human Research Ethics Committee of the University of Sydney. Informed consent was obtained from the donors for this study.
1. Human RBC isolation
NOTE: Step 1.1 should be performed by a trained phlebotomist using a protocol that has been approved by the Institutional Review Board.
<ol>
	<li>Withdraw 5 mL of blood from the median cubital vein using a 19 G butterfly needle.</li>
	<li>Transfer the collected blood into a 15 mL tube containing 1:200 enoxaparin to prevent clotting.</li>
	<li>Dilute 5 &#181;L of enoxaparin-anticoagulated blood in 1 mL of carbonate/bicarbonate buffer (C-buffer, pH = 8.5-9; Table of Materials).</li>
	<li>Centrifuge the diluted blood sample at 900 &#215; g for 1 min to sediment the RBCs. Carefully decant the supernatant without disturbing the pellet.</li>
	<li>Perform two washes of the RBC pellet with 1 mL of C-buffer (Table of Materials), centrifuging each time at 900 &#215; g for 1 min.</li>
	<li>Subsequently, wash the RBC pellet 2x with 1 mL of Tyrode&#39;s buffer using the same centrifugation conditions and then resuspend the final pellet in 1 mL of Tyrode&#39;s buffer to obtain the washed stock RBC suspension.</li>
</ol>
2. Calcium indicator loading
<ol>
	<li>Adjust the concentration of the washed stock RBC solution to 10 &#215; 106 cells/mL in Tyrode&#39;s buffer, based on the cell count obtained using an automatic cell counter (Table of Materials).</li>
	<li>Label the calcium inside the RBCs by incubating with 16.67 &#181;M Cal-520 AM, a calcium-sensitive dye, while agitating on a rotary tube mixer for 1 h.</li>
	<li>Dilute the RBCs in Tyrode&#39;s buffer containing 0.5% bovine serum albumin (BSA) at a 1:50 ratio. The cells are now ready for experimental use.</li>
</ol>
3. Micropipette fabrication
<ol>
	<li>Mount the borosilicate glass capillary tube (1 mm outer diameter x 0.6 mm inner diameter) onto the P-1000 micropipette puller to produce two corresponding micropipettes with closed tips at the pulling site using the preset pulling program. For this setup, use the following pulling program values: heat 516, pull 150, velocity 75, time 250, and pressure 500. 
	NOTE: Heating and pulling parameters set in the pulling program can be customized and are dependent on the desired settings of the experimental design12. CHECKPOINT (see Supplemental Table S1).</li>
	<li>Open the closed tip by mounting one of the close-ended micropipettes procured after pulling onto the micropipette cutter. Adjust the heating temperature to approximately 50-60 &#176;C.</li>
	<li>Locate the micropipette using a 10x eyepiece. Move the micropipette close to the borosilicate glass bead by using the knobs for adjustment.</li>
	<li>Change the eyepiece to 30x before positioning the micropipette as close to the borosilicate glass bead as possible without bending the pipette tip.</li>
	<li>Soften the borosilicate glass bead using heat by stepping onto the heating pedal. Gently insert the raw closed micropipette tip into the softened bead until the desired endpoint,&#160;the opening diameter, has been reached.</li>
	<li>Release the foot pedal&#160;and let the glass bead cool down. Make sure the tip of the micropipette always remains inside the bead. 
	NOTE: Inserting the tip further leads to larger opening diameters.</li>
	<li>Gently extract the micropipette, leading to a clear straight cut on the closed micropipette. Confirm that the final diameter of the capillary is 1 &#181;m. 
	&#8203;NOTE: CHECKPOINT (see Supplemental Table S1)</li>
</ol>
4. Cell chamber preparation
<ol>
	<li>Use a diamond pencil to divide a standard 40 mm x 22 mm x 0.17 mm glass coverslip into three equal strips.</li>
	<li>Adhere one piece of the cut glass coverslip to the bottom of a homemade chamber holder with vacuum grease. 
	NOTE: The chamber holder consists of two metal (copper/aluminum) squares that are linked by a curved handle. The distance between the metal blocks must span less than 40 mm for the cut coverslip to adhere to the holder to form a parallel chamber.</li>
	<li>Adhere the second piece of the cut glass coverslip to the top of the homemade chamber holder with vacuum grease . 
	NOTE: CHECKPOINT (see Supplemental Table S1)</li>
	<li>Inject 200 &#181;L of the labeled RBC suspension between two coverslips using a 200 &#181;L pipette gun (Figure 1).</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66265/66265fig01.jpg" /> 
Figure 1: Illustration of the cell chamber. Two cut pieces of a 40 mm x 22 mm x 0.17 mm glass cover slip are adhered to the chamber holder using grease. Between the two cut glass coverslips, approximately 200 &#181;L of the cell solution in Tyrode&#39;s Buffer is seeded. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66265/66265fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
5. Micropipette aspiration assembly
<ol>
	<li>Mount the cell chamber onto the holder stage present on the microscope platform. Adjust the position so that the cell chamber is directly above the objective (Figure 2B).</li>
	<li>Lower the micropipette holder to below the fluid level of the connected water reservoir.</li>
	<li>Inject either demineralized water or Tyrode&#39;s buffer into the fabricated micropipette and carefully remove all air bubbles using a syringe coupled with a 34 G needle (see Table of Materials).</li>
	<li>Unscrew the end of the micropipette holder halfway and allow the water to drip from the micropipette holder for a few seconds. 
	NOTE: CHECKPOINT (see Supplemental Table S1)</li>
	<li>Insert the&#160;micropipette into the holder tip. Tighten the holder screw to ensure the micropipette is fixed.</li>
	<li>Insert the micropipette into the cell chamber and locate the micropipette and RBCs under the microscope. Use the micromanipulator to adjust the position.</li>
	<li>Lower the micropipette tip further to ensure the tip is leveled with the located RBC. 
	NOTE: CHECKPOINT (see Supplemental Table S1)</li>
	<li>Zero the hydraulic pressure at the micropipette tip by adjusting the height of the water reservoir. Then, slightly raise the water reservoir to generate a subtle positive pressure at the tip.</li>
</ol>
6. Perform the fluorescence-coupled micropipette aspiration assay
<ol>
	<li>Turn on the 488 nm fluorescent excitation light source. Do not switch on the fluorescence shutter at this stage to avoid photobleaching (Figure 2C). Turn on the fluorescence camera and the transmitted camera. 
	NOTE: Both cameras are operated using the appropriate software (see Table of Materials).</li>
	<li>Set up the desired exposure time (100 ms for both cameras in this study), region of interest (ROI), binning size (none for this study) for both cameras in the software. Open up the multi-dimension acquisition panel to set up the acquisition frame number, 2,000 for this study, and saving directory. 
	NOTE: The acquisition frame number is dependent on the desired number of aspiration events that are to be recorded. For 1 aspiration event, the range of the acquisition number should be set within 100-500, which is approximately 10-50 s.</li>
	<li>Find the micropipette under the field of view using the micromanipulator.</li>
	<li>Turn on the pneumatic pressure clamp, including the control box and the clamp system (Figure 2A). Make sure the control box is in the EXTRNL mode. Compensate any offset pressure inside the system by slowly rotating the knob.</li>
	<li>Turn on the separate software that controls the pneumatic clamp. The software has an electrical control panel to control the discrete analog input to the clamp system. The pressure is controlled with a 20 mV/mmHg conversion factor.</li>
	<li>Zero the pressure inside the system. Carefully relocate the micropipette close to the RBCs. Adjust the water reservoir position until a subtle positive pressure is noticed at the micropipette tip.</li>
	<li>Start the acquisition in the camera-operating software. Switch on the fluorescence shutter.</li>
	<li>Aspirate an RBC by typing in the calculated voltage magnitude into the control panel to reach the desired pressure. 
	NOTE: The pressure for aspirating an RBC is typically in the range of &#916;p = -5 to -40 mmHg. There should be a noticeable tongue elongation within the micropipette tip (Figure 2D).</li>
	<li>Hold the pressure for a preset period; then, release the pressure.</li>
	<li>Move the micropipette to pick up the next cell and repeat the experiment.</li>
</ol>
7. Fluorescence intensity analysis
<ol>
	<li>Load the saved fluorescence images into the analysis software.</li>
	<li>Adjust the intensity threshold using the display adjustment tab. Do this by either manually inputting the values or using the slider to ensure the fluorescence images show a clear contrast of the cell in the analysis software (see Supplemental File 1-Supplemental Figure S1).</li>
	<li>Scroll to the timeline at the bottom of the software. Locate the designated aspiration event.</li>
	<li>Click Add new surfaces. Define the analysis ROI. 
	NOTE: The software provides a guided five-step process to adjust and complete the segmentation (see Supplemental File 1-Supplemental Figure S2 and Supplemental Figure S3). 
	NOTE: Keep the ROI as small as possible to save computational resources.</li>
	<li>Use the background subtraction slider and adjust the segmentation threshold using the slider to obtain the best segmentation outcome. 
	NOTE: This means that apart from the aspiration event, the background should be segmented as accurately as possible (see Supplemental File 1-Supplemental Figure S4 and Supplemental Figure S5).</li>
	<li>Add an area filter to exclude background noises (see Supplemental File 1-Supplemental Figure S6). 
	NOTE: This is completed in the post process stage.</li>
	<li>Select the statistics tab | detailed tab | average values tab. Scroll to find and select the intensity mean (see Supplemental File 1-Supplemental Figure S7).</li>
	<li>Export the fluorescence signal trace over time to a .csv file.</li>
	<li>Open the exported csv file. Subtract the background signals, Fb, from all measurements.</li>
	<li>Calculate the calcium intensity change, &#916;Fmax, using equation (1): 
	<img alt="Equation 1" src="/files/ftp_upload/66265/66265eq01.jpg" style="margin: auto;" />&#160; &#160; &#160;(1) 
	Where&#160;&#916;Fmax is the maximum calcium intensity change, Fb, is the background intensity, and F0 is the resting intensity.</li>
</ol>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66265/66265fig02.jpg" /> 
Figure 2: Fluorescence-coupled micropipette aspiration assembly. (A)&#160;An overview of the fMPA hardware system incorporating the inverted microscope combined with the brightfield and fluorescence cameras. The left side of the image depicts the homemade water manometer and the control box that allows to precisely tune the pressure of the pneumatic pressure pump. (B)&#160;The microscope stage depicting the experiment cell chamber and micromanipulator system with a single micropipette. (C)&#160;Schematic of the fMPA system setup. Concurrent imaging of brightfield (yellow) and fluorescence (blue emission, green excitation) signals utilizing two dichroic mirrors to direct the light paths from the fluorescence light source (blue) to the target, then to the cameras for imaging (green). (D)&#160;The top row depicts the brightfield images whereas the bottom row demonstrates the fluorescence images. The left represents the position of the micropipette before aspiration when the RBC&#160;is at rest.The middle column snapshots the aspiration process where the RBC experiences a negative pressure of -40 mmHg. The right depicts the cell morphology after experiencing the negative aspiration pressure. Scale bar = 5 &#181;m. Abbreviations: fMPA = Fluorescence-coupled Micropipette Aspiration; DM = dichroic mirror; RBC = red blood cell. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66265/66265fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Single-Cell Mechanobiology Using Fluorescence Micropipette Aspiration

<ol>
	<li>Gonz&#225;lez-Berm&#250;dez, B., Guinea, G. V., Plaza, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+micropipette+aspiration:+applications+in+cell+biomechanics,+models,+and+extended+studies.">Advances in micropipette aspiration: applications in cell biomechanics, models, and extended studies.</a> Biophysical Journal. 116 (4), 587-594 (2019).</li><li>Mierke, C. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Physics of Cancer, Volume 3 (Second Edition): Experimental biophysical techniques in cancer research. , (2021).</li><li>Chen, Y., 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=Loss+of+the+F-BAR+protein+CIP4+reduces+platelet+production+by+impairing+membrane-cytoskeleton+remodeling.">Loss of the F-BAR protein CIP4 reduces platelet production by impairing membrane-cytoskeleton remodeling.</a> Blood. 122 (10), 1695-1706 (2013).</li><li>Shin, J. -. W., Swift, J., Spinler, K. R., Discher, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myosin-II+inhibition+and+soft+2D+matrix+maximize+multinucleation+and+cellular+projections+typical+of+platelet-producing+megakaryocytes.">Myosin-II inhibition and soft 2D matrix maximize multinucleation and cellular projections typical of platelet-producing megakaryocytes.</a> Proceedings of the National Academy of Sciences. 108 (28), 11458-11463 (2011).</li><li>Liapis, H., Foster, K., Miner, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Red+cell+traverse+through+thin+glomerular+basement+membrane.">Red cell traverse through thin glomerular basement membrane.</a> Kidney International. 61 (2), 762-763 (2002).</li><li>Wang, H., 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=Fluorescence-coupled+micropipette+aspiration+assay+to+examine+calcium+mobilization+caused+by+red+blood+cell+mechanosensing.">Fluorescence-coupled micropipette aspiration assay to examine calcium mobilization caused by red blood cell mechanosensing.</a> European Biophysics Journal. 51 (2), 135-146 (2022).</li><li>Danielczok, J. G., 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=Red+blood+cell+passage+of+small+capillaries+is+associated+with+transient+Ca2+-mediated+adaptations.">Red blood cell passage of small capillaries is associated with transient Ca2+-mediated adaptations.</a> Frontiers in Physiology. 8, 979 (2017).</li><li>Diez-Silva, M., Dao, M., Han, J., Lim, C. -. T., Suresh, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shape+and+biomechanical+characteristics+of+human+red+blood+cells+in+health+and+disease.">Shape and biomechanical characteristics of human red blood cells in health and disease.</a> MRS Bulletin. 35 (5), 382-388 (2010).</li><li>Ma&#238;tre, J. -. L., Niwayama, R., Turlier, H., N&#233;d&#233;lec, F., Hiiragi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pulsatile+cell-autonomous+contractility+drives+compaction+in+the+mouse+embryo.">Pulsatile cell-autonomous contractility drives compaction in the mouse embryo.</a> Nature Cell Biology. 17 (7), 849-855 (2015).</li><li>Ju, L., Chen, Y., Xue, L., Du, X., Zhu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cooperative+unfolding+of+distinctive+mechanoreceptor+domains+transduces+force+into+signals.">Cooperative unfolding of distinctive mechanoreceptor domains transduces force into signals.</a> eLife. 5, e15447 (2016).</li><li>Bogdanova, A., Makhro, A., Wang, J., Lipp, P., Kaestner, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+in+red+blood+cells-a+perilous+balance.">Calcium in red blood cells-a perilous balance.</a> International Journal of Molecular Sciences. 14 (5), 9848-9872 (2013).</li><li>. Pipette Cookbook 2018 Available from: <a target="_blank" href="https://www.sutter.com/PDFs/cookbook.pdf">https://www.sutter.com/PDFs/cookbook.pdf</a> (2018)</li><li>Cahalan, S. M., 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=Piezo1+links+mechanical+forces+to+red+blood+cell+volume.">Piezo1 links mechanical forces to red blood cell volume.</a> eLife. 4, e07370 (2015).</li><li>Sforna, L., 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=Piezo1+controls+cell+volume+and+migration+by+modulating+swelling-activated+chloride+current+through+Ca2++influx.">Piezo1 controls cell volume and migration by modulating swelling-activated chloride current through Ca2+ influx.</a> Journal of Cellular Physiology. 237 (3), 1857-1870 (2022).</li><li>High speed pressure clamp. ALA Scientific Instruments Available from: <a target="_blank" href="https://alascience.com/products/hspc-2sb/">https://alascience.com/products/hspc-2sb/</a> (2023)</li><li>. Teledyne Imaging Prime 95BTM Scientific CMOS Camera Datasheet Available from: <a target="_blank" href="https://www.photometrics.com/wp-content/uploads/2019/10/Prime-95B-Datasheet-07172020.pdf">https://www.photometrics.com/wp-content/uploads/2019/10/Prime-95B-Datasheet-07172020.pdf</a> (2020)</li><li>Lee, L. M., Lee, J. W., Chase, D., Gebrezgiabhier, D., Liu, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+an+advanced+microfluidic+micropipette+aspiration+device+for+single+cell+mechanics+studies.">Development of an advanced microfluidic micropipette aspiration device for single cell mechanics studies.</a> Biomicrofluidics. 10 (5), 054105 (2016).</li><li>Weaver, W. M., 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=Advances+in+high-throughput+single-cell+microtechnologies.">Advances in high-throughput single-cell microtechnologies.</a> Current Opinion in Biotechnology. 25, 114-123 (2014).</li><li>Zhou, E. H., Lim, C. T., Quek, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Finite+element+simulation+of+the+micropipette+aspiration+of+a+living+cell+undergoing+large+viscoelastic+deformation.">Finite element simulation of the micropipette aspiration of a living cell undergoing large viscoelastic deformation.</a> Mechanics of Advanced Materials and Structures. 12 (6), 501-512 (2005).</li><li>Oh, M. -. J., Kuhr, F., Byfield, F., Levitan, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micropipette+aspiration+of+substrate-attached+cells+to+estimate+cell+stiffness.">Micropipette aspiration of substrate-attached cells to estimate cell stiffness.</a> Journal of Visualized Experiments. (67), e3886 (2012).</li><li>Rand, R. P., Burton, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+properties+of+the+red+cell+membrane.">Mechanical properties of the red cell membrane.</a> Biophysical journal. 4 (4), 303-316 (1964).</li><li>Hogan, B., Babataheri, A., Hwang, Y., Barakat, A. I., Husson, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterizing+cell+adhesion+by+using+micropipette+aspiration.">Characterizing cell adhesion by using micropipette aspiration.</a> Biophysical Journal. 109 (2), 209-219 (2015).</li><li>Henriksen, J. R., Ipsen, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+membrane+elasticity+by+micro-pipette+aspiration.">Measurement of membrane elasticity by micro-pipette aspiration.</a> The European Physical Journal E. 14 (2), 149-167 (2004).</li><li>Hochmuth, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micropipette+aspiration+of+living+cells.">Micropipette aspiration of living cells.</a> Journal of Biomechanics. 33 (1), 15-22 (2000).</li><li>Pu, H., 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=Micropipette+aspiration+of+single+cells+for+both+mechanical+and+electrical+characterization.">Micropipette aspiration of single cells for both mechanical and electrical characterization.</a> IEEE Transactions on Biomedical Engineering. 66 (11), 3185-3191 (2019).</li><li>Guevorkian, K., Ma&#238;tre, J. -. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+10+-+Micropipette+aspiration:+A+unique+tool+for+exploring+cell+and+tissue+mechanics+in+vivo.">Chapter 10 - Micropipette aspiration: A unique tool for exploring cell and tissue mechanics in vivo.</a> Methods in Cell Biology. , 187-201 (2017).</li><li>Biro, M., Ma&#238;tre, J. -. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+14+-+Dual+pipette+aspiration:+A+unique+tool+for+studying+intercellular+adhesion.">Chapter 14 - Dual pipette aspiration: A unique tool for studying intercellular adhesion.</a> Methods in Cell Biology. 125, 255-267 (2015).</li></ol>

The authors declare that they have no competing interests to report regarding the present study.

Micropipette aspiration assays embody a refined methodology, deploying substantial pressure modulation, exact spatial orchestration, and reliable temporal discernment to probe the profound intricacies of cellular biomechanics. This study places particular emphasis on the application of fMPA as a crucial tool for unveiling the nuanced mechanosensitive responses showcased by RBCs under varying stimuli. The concurrent use of brightfield and fluorescence signals enabled a multifaceted exploration of cellular phenomena, advancing the monitoring and detection of intracellular calcium influx in real time. This approach provides an integrative insight into the complex mechanosensing reactions of RBCs.
Importantly, the applicability of the fMPA technique extends beyond RBCs, as it can be employed with other cell types that disapply high mechanical sensitivity, such as platelets and neurons9. Moreover, owing to its minimal impact on cell integrity during experimental handling, fMPA guarantees the preservation of the cell&#39;s natural state, making it highly suitable for use with primary cells1. Furthermore, the fMPA method offers versatility through the tunable geometry of the micropipettes, allowing for a broader range of experimental designs tailored to specific research questions and the effective exploration of various mechanical conditions.
Additionally, with the improvement in the optical pathway that enhances the simultaneous use of multi-fluorescence imaging, the fMPA system allows for real-time detection of intracellular calcium concentration upon aspiration. This capability opens an opportunity to explore calcium-related molecules, such as the mechanosensitive ion channel, PIEZO1, which has been shown to mediate and perceive mechanical cues from the external environments7,14. Recent literature highlights an increase in membrane tension as a significant factor stimulating PIEZO1 channel activity, subsequently facilitating the Ca2+ influx6. This underscores a crucial application of fMPA, which is to investigate the interplay between membrane tension, PIEZO1, and calcium influx, shedding light on the intricate mechanosensing processes within cells.
From a technical perspective, it is worth mentioning that the two primary components of the fMPA system, responsible for generating the aspiration force (mechanical stimuli) and conducting real-time fluorescence imaging, are the pneumatic pressure pump and the fluorescence camera, respectively. The selection of the appropriate pump and camera for the system should be based on the specific cell type and biological process under study. The pump employed in our system operates within a steady-state pressure range of &#177; 200 mmHg and can respond to commands for pressure changes as significant as &#177; 200 mmHg. The pressure rise time and fall time should not exceed 6-8 ms for a 20 mmHg step and 15-20 ms for a 200 mmHg step. To ensure force resolution, the noise level of the HSPC system should be less than &#177; 0.5 mmHg, peak-to-peak. These requirements are applied in this setup; however, a range of &#177; 40 mmHg is sufficient to achieve a response for the experimental protocol15. Regarding the fluorescence camera, we selected features a 95% Quantum Efficiency and an 11 &#181;m x 11 &#181;m pixel size chip. This sensitive sensor configuration efficiently captures the fluorescence signal at high speed. Additionally, it is crucial to maintain a median read noise of 1.6e- for an adequate signal-to-noise ratio during imaging16.
From a procedural perspective, executing fMPA requires a set of advanced skills. For example, crafting micropipettes using the micropipette cutter demands precision and dexterity, along with meticulous control of temperature and positioning. Positioning&#160;the micropipette within the microscope&#39;s field of view&#160;requires meticulous effort to avoid damage to both the micropipette tip and the cellular chamber. Additionally, one common challenge associated with fluorescence imaging is photobleaching. To mitigate this issue, it is important to minimize the sample&#39;s exposure time to the illumination system. With reference to the &#39;fluorescence-coupled micropipette aspiration assay&#39; protocol, the fluorescence shutter of the excitation light source remains switched off until all parameters are inputted through the camera-operating software and the aspiration system is ready for experiments. Only then is the fluorescence shutter turned on to commence imaging. To further reduce the photobleaching, it is recommended to use the minimum light source intensity that still yields adequate fluorescence expression in the sample.
Furthermore, the current manual operation required for fMPA assays makes this technique labor-intensive. Consequentially, the inconsistencies that may arise in this assay primarily stem from operator-dependent variables, as well as the factors associated with variations in filament preheating and capillary glass characteristics. In the future, optimizing the performance of this technique necessitates consideration of potential tandem implementations. For example, when combined with microfluidic devices, the micropipette aspiration can be transformed into a high-throughput platform, significantly increasing the experimental rate from studying around 20 cells per hour to approximately 1,000 cells/h17,18. This incorporation also improves the reproducibility of the data. Moreover, certain avenues have been successfully explored by incorporating automation and image analysis systems19. Notably, finite element analysis (FEA) is a computational tool commonly employed to model micropipette aspiration assays. FEA can predict the cellular response to mechanical stimuli and characterize their mechanical properties. It holds the potential to optimize the micropipette design and further validate the experimental results19.
In conclusion, the fMPA approach offers valuable insights into the mechanosensitive behaviors of RBCs in responding to mechanical stimuli. This study establishes a foundational framework for future investigations into the intricate mechanisms of mechanotransduction within RBCs and across broader biological systems. Such inquiries hold great promise for advancing our understanding of these mechanisms and unraveling their extensive implications in various physiological contexts.

The unfolding discoveries in the world of cellular behaviors have accentuated the role of mechanical stimuli, such as tension, fluid shear stress, compression, and substrate stiffness, in dictating dynamic cellular activities such as adhesion, migration, and differentiation. These mechanobiological aspects are of paramount importance in elucidating how cells interact with and respond to their physiological environments, impacting various biological processes1,2.
Over the past decade, micropipette-based aspiration assays have stood out as a versatile tool in studying diverse cellular responses to mechanical stimuli. This technique offers valuable insights into the intrinsic mechanical properties of living cells at the single-cell level, including cellular elastic modulus, stiffness, and cortical&#160;tension. These assays enable the measurement of various mechanical parameters, such as cell membrane&#160;tension, pressure exerted on the cell membrane, and cortical tension (summarized in Table 1). Studying the aspirational forces has enriched our understanding of how they influence cellular functions and processes, particularly in the realm of membrane dynamics, including fragmentation, elongation, and budding3,4.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Mechanical Parameter</td>
			<td>Description</td>
			<td>Seminal Approaches</td>
		</tr>
		<tr>
			<td>Cell Stiffness</td>
			<td>Measurement of a cell&#39;s mechanical rigidity and elasticity.</td>
			<td>Aspiration of the cell membrane and analysis of deformation response to the negative pressure20,21.</td>
		</tr>
		<tr>
			<td>Adhesion Strength</td>
			<td>Evaluation of how strongly cells adhere to surfaces.</td>
			<td>Application of controlled suction to detach adhered cells from a substrate2,22.</td>
		</tr>
		<tr>
			<td>Membrane Tension</td>
			<td>Assessment of the tension or stress within cell membranes.</td>
			<td>Measurement of the membrane deformation in response to applied pressure23,24.</td>
		</tr>
		<tr>
			<td>Viscoelastic Properties</td>
			<td>Characterization of a cell&#39;s combined viscous and elastic behavior.</td>
			<td>Analysis of the time-dependent deformation response to aspiration23,25.</td>
		</tr>
		<tr>
			<td>Deformability</td>
			<td>Determination of how easily a cell can change shape.</td>
			<td>Evaluation of the extent of deformation under controlled suction20,24.</td>
		</tr>
		<tr>
			<td>Surface Tension</td>
			<td>Measurement of the tension at the cell&#39;s surface.</td>
			<td>Assessment of the pressure required to form a micropipette membrane protrusion26.</td>
		</tr>
		<tr>
			<td>Cell-Material Interaction</td>
			<td>Study of interactions between cells and materials or substrates.</td>
			<td>Aspiration of cells in contact with different materials and observation of interactions2,24.</td>
		</tr>
		<tr>
			<td>Cell-Cell Interaction</td>
			<td>Examination of interactions between neighboring cells.</td>
			<td>Aspiration of a group of cells and analysis of their intercellular forces27.</td>
		</tr>
	</tbody>
</table>
Table 1: Mechanical parameters characterized by the micropipette aspiration assay.
The micropipette-based aspiration technique has been widely used to study red blood cells (RBCs), assessing the deformability and various mechanical characteristics of RBCs, which is essential in understanding their function in the circulatory system. RBCs exhibit remarkable adaptability, preserving their mechanical versatility against deformation when navigating through the intricate capillary network and inter-endothelial clefts5,6. During this journey, RBCs must traverse through passages as narrow as 0.5-1.0 &#181;m, subjecting themselves to a multitude of mechanical forces, including tension and compression7,8,9. They also have high sensitivity to the shear stress generated by blood flow during circulation10. These processes promote the activation of regulatory mechanisms involving calcium influx, a crucial signaling event with well-established roles in cellular responses to mechanical stimuli11,12. The complex mechanisms governing the calcium-mediated mechanosensing remain compelling subjects of ongoing investigation.
In this context, the fMPA stands as an effective approach to reveal the extent of calcium mobilization under precisely controlled mechanical forces, allowing for the simultaneous application of mechanical modulation (using the micropipette aspiration system) and visualization of calcium intensity (using fluorescent indicators). It particularly mimics the physiological scenario when the RBC travels through narrowing blood vessels. It is worth noting that the fMPA system we developed can generate pressure with a resolution of 1 mmHg. The implemented high-speed camera can achieve a temporal resolution of 100 ms and a spatial resolution at the submicron-meter level. These configurations ensure the precise application of mechanical forces to live cells and simultaneously capture the resulting cellular signaling. Moreover, due to the integrative engineered nature of this setup, the micropipette aspiration assay can be readily adapted to complement other equipment or techniques, enabling further exploration of the intricacies of cell mechanics. This versatility stands as an additional advantage of this approach.

<table><tbody><tr><td>&amp;micro;Manager</td><td>Micro-Manager</td><td></td><td>Version 2.0.0</td></tr><tr><td>1 mL Syringe&amp;nbsp;</td><td>Terumo</td><td>210320D</td><td>Cooperate with the Microfil&amp;nbsp;</td></tr><tr><td>200 &amp;micro;L Pipette&amp;nbsp;</td><td>Eppendorf&amp;nbsp;</td><td>3123000055</td><td>Red clood cell preparation</td></tr><tr><td>22 x 40 mm Cover Slips</td><td>Knittel Glass&amp;nbsp;</td><td>MS0014</td><td>Cell chamber assembly</td></tr><tr><td>50 mL Syringe&amp;nbsp;</td><td>Terumo</td><td>220617E</td><td>Connect to the water tower</td></tr><tr><td>Calcium Chloride (CaCl&lt;sub&gt;2&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>C1016</td><td>Tryode&#039;s&amp;nbsp; buffer preparation - 12 mM NaHCO&lt;sub&gt;3&lt;/sub&gt;, 10 mM HEPES, 0.137 M NaCl, 2.7 mM KCl, and 5.5 mM D-glucose supplemented with 1 mM CaCl&lt;sub&gt;2&lt;/sub&gt;. Final pH = 7.2</td></tr><tr><td>Centrifuge 5425</td><td>Eppendorf&amp;nbsp;</td><td>5405000280</td><td>Red clood cell preparation</td></tr><tr><td>Clexane</td><td>Sigma-Aldrich</td><td>1235820</td><td>To prevent clotting of the collected blood. 10,000 U/mL</td></tr><tr><td>DAQami</td><td>Diligent</td><td></td><td></td></tr><tr><td>Fluorescence light source</td><td>CoolLED</td><td>pE-300</td><td>Micropipette aspiration hardware system</td></tr><tr><td>Glass capillary</td><td>Narishige</td><td>G-1</td><td>Micropipette manufacture</td></tr><tr><td>Glucose</td><td>Sigma-Aldrich</td><td>G8270</td><td>Tryode&#039;s&amp;nbsp; buffer preparation - 12 mM NaHCO&lt;sub&gt;3&lt;/sub&gt;, 10 mM HEPES, 0.137 M NaCl, 2.7 mM KCl, and 5.5 mM D-glucose supplemented with 1 mM CaCl&lt;sub&gt;2&lt;/sub&gt;. Final pH = 7.2</td></tr><tr><td>Hepes</td><td>Thermo Fisher</td><td>15630080</td><td>Tryode&#039;s&amp;nbsp; buffer preparation - 12 mM NaHCO&lt;sub&gt;3&lt;/sub&gt;, 10 mM HEPES, 0.137 M NaCl, 2.7 mM KCl, and 5.5 mM D-glucose supplemented with 1 mM CaCl&lt;sub&gt;2&lt;/sub&gt;. Final pH = 7.2</td></tr><tr><td>High speed GigE camera</td><td>Manta</td><td>G-040B</td><td>Micropipette aspiration hardware system</td></tr><tr><td>High speed pressure clamp</td><td>Scientific Instrument</td><td>HSPC-2-SB</td><td>Cooperate with the pressure pump</td></tr><tr><td>High speed pressure clamp head stage&amp;nbsp;</td><td>Scientific Instrument</td><td>HSPC-2-SB</td><td>Cooperate with the pressure pump</td></tr><tr><td>Imaris</td><td>Oxford Instruments</td><td></td><td></td></tr><tr><td>Inverted Microscopy&amp;nbsp;</td><td>Olympus&amp;nbsp;</td><td>Olympus IX83</td><td>Micropipette aspiration hardware system</td></tr><tr><td>Microfil&amp;nbsp;</td><td>World Precision Instruments&amp;nbsp;</td><td>MF34G-5</td><td>34 G (67 mm Long)&lt;br/&gt; Revome air bubble in the cut micropipette and test the opening of the pipette tip&amp;nbsp;</td></tr><tr><td>Micropipette Puller&amp;nbsp;</td><td>Sutter instrument</td><td>P1000</td><td>Micropipette manufacture&amp;nbsp;</td></tr><tr><td>Milli Q EQ 7000 Ultrapure Water Purification System</td><td>Merck Millipore</td><td>ZEQ7000T0C</td><td>Carbonate/bicarbonate buffer &amp;amp; Tryode&#039;s buffer preparation</td></tr><tr><td>Pipette microforge&amp;nbsp;</td><td>Narishige</td><td>MF-900</td><td>Micropipette manufacture</td></tr><tr><td>Potassium Chloride (KCl)</td><td>Sigma-Aldrich</td><td>P9541</td><td>Tryode&#039;s&amp;nbsp; buffer preparation - 12 mM NaHCO&lt;sub&gt;3&lt;/sub&gt;, 10 mM HEPES, 0.137 M NaCl, 2.7 mM KCl, and 5.5 mM D-glucose supplemented with 1 mM CaCl&lt;sub&gt;2&lt;/sub&gt;. Final pH = 7.2</td></tr><tr><td>Pressue Pump&amp;nbsp;</td><td>Scientific Instrument</td><td>PV-PUMP</td><td>Induce controlled pressure during experiment</td></tr><tr><td>Prime 95B Camera&amp;nbsp;</td><td>Photometrics</td><td>Prime 95B sCMOS</td><td>Flourscent imaging</td></tr><tr><td>Rotary wheel remote unit&amp;nbsp;</td><td>Sensapex&amp;nbsp;</td><td>uM-RM3</td><td>Control panel for micropipette position adjustment&amp;nbsp;</td></tr><tr><td>Scepter 3.0 Handheld Cell Counter</td><td>Merck Millipore</td><td>PHCC340KIT</td><td>Automatic cell counter</td></tr><tr><td>Sodium Bicarbonate (NaHCO&lt;sub&gt;3&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>S5761</td><td>Carbonate/bicarbonate buffer preparation - 2.65 g of NaHCO&lt;sub&gt;3&lt;/sub&gt; with 2.1 g of Na&lt;sub&gt;2&lt;/sub&gt;CO&lt;sub&gt;3&lt;/sub&gt; in 250 mL of Mili Q water - Final pH = 8-9.</td></tr><tr><td>Sodium Carbonate (Na&lt;sub&gt;2&lt;/sub&gt;CO&lt;sub&gt;3&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>S2127</td><td>Carbonate/bicarbonate buffer preparation - 2.65 g of NaHCO&lt;sub&gt;3&lt;/sub&gt; with 2.1 g of Na&lt;sub&gt;2&lt;/sub&gt;CO&lt;sub&gt;3&lt;/sub&gt; in 250 mL of Mili Q water - Final pH = 8-9.</td></tr><tr><td>Sodium Chloride (NaCl)</td><td>Sigma-Aldrich</td><td>S7653</td><td>Tryode&#039;s&amp;nbsp; buffer preparation - 12 mM NaHCO&lt;sub&gt;3&lt;/sub&gt;, 10 mM HEPES, 0.137 M NaCl, 2.7 mM KCl, and 5.5 mM D-glucose supplemented with 1 mM CaCl&lt;sub&gt;2&lt;/sub&gt;. Final pH = 7.2</td></tr><tr><td>Sodium Phosphate Monobasic&lt;br/&gt; Monohydrate (NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt; &amp;bull; H&lt;sub&gt;2&lt;/sub&gt;O)</td><td>Sigma-Aldrich</td><td>S9638</td><td>Tryode&#039;s&amp;nbsp; buffer preparation - 12 mM NaHCO&lt;sub&gt;3&lt;/sub&gt;, 10 mM HEPES, 0.137 M NaCl, 2.7 mM KCl, and 5.5 mM D-glucose supplemented with 1 mM CaCl&lt;sub&gt;2&lt;/sub&gt;. Final pH = 7.2</td></tr><tr><td>Touch screen control unit&amp;nbsp;</td><td>Sensapex&amp;nbsp;</td><td>uM-TSC</td><td>Control panel for micropipette position adjustment&amp;nbsp;</td></tr><tr><td>X dry Objective&amp;nbsp;</td><td>Olympus&amp;nbsp;</td><td>Olympus 60x/0.70 LUCPlanFL</td><td>Micropipette aspiration hardware system</td></tr></tbody></table>

fluorescence micropipette aspiration assay to investigate red blood cell mechanosensing

Micropipette aspiration assays have long been a cornerstone for the investigation of live-cell mechanics, offering insights into cellular responses to mechanical stress. This paper details an innovative adaptation of the fluorescence-coupled micropipette aspiration (fMPA) assay. The fMPA assay introduces the capability to administer precise mechanical forces while concurrently monitoring the live-cell mechanotransduction processes mediated by ion channels. The sophisticated setup incorporates a precision-engineered borosilicate glass micropipette connected to a finely regulated water reservoir and pneumatic aspiration system, facilitating controlled pressure application with increments as refined as &#177; 1 mmHg. A significant enhancement is the integration of epi-fluorescence imaging, allowing for the simultaneous observation and quantification of cell morphological changes and intracellular calcium fluxes during aspiration. The fMPA assay, through its synergistic combination of epi-fluorescence imaging with micropipette aspiration, sets a new standard for the study of cell mechanosensing within mechanically challenging environments. This multifaceted approach is adaptable to various experimental setups, providing critical insights into the single-cell mechanosensing mechanisms.

To establish micropipette aspiration assays, we first constructed a custom cell chamber comprising two metal squares (copper/aluminum) connected by a handle. Two third-cut glass coverslips (40 mm &#215; 7 mm &#215; 0.17 mm) were affixed to create a chamber filled with 200 &#181;L of RBCs suspended in Tyrode&#39;s Buffer. After introducing RBCs into the chamber, a tailored borosilicate micropipette was secured on a holder and carefully positioned within the chamber using a micro-manipulator. Subsequently, the micropipette was brought closer to capture the target RBC.
For cell aspiration, the method used a pneumatic high-speed pressure clamp to fine-tune the negative aspiration pressure. Fluorescence imaging was then conducted to investigate the calcium mobilization at the different negative aspiration pressures applied.
By using the fMPA to investigate how RBCs respond to varying negative aspiration pressures, our findings reveal that there is a clear proportional relationship between the negative pressure applied and the calcium influx present in the aspirated RBC. To determine the calcium intensity change, the maximum intensity of the single aspirated cell (Fmax) minus the background intensity (Fb) was divided by the resting intensity (F0) minus Fb. There was a corresponding increase in the influx of calcium ions into the RBCs when the pressure was incrementally increased between -10 mmHg (Figure 3A) to -40 mmHg (Figure 3D). This suggests that RBCs possess the ability to sense changes in their mechanical and respond by rapid calcium-specific channel activities14,15.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/66265/66265fig03.jpg" /> 
Figure 3: Fluorescence&#160;imaging with a graphical representation of normalized intensity changes against time. Horizontally across depicts&#160;fluorescence snapshots of the RBCs at rest (left) and&#160;the human RBCs being aspirated by the micropipette (middle).&#160;Representative traces of the RBCs being aspirated at multiple negative aspiration pressures (&#916;p) can be seen on the right. The negative aspiration pressure is incrementally increased, starting at (A) &#916;p = -10 mmHg, (B)&#160;&#916;p = -20 mmHg, (C)&#160;&#916;p = -30 mmHg, and (D)&#160;&#916;p = -40 mmHg. When the tongue of the RBC is elongated during aspiration, a significant calcium mobilization can be observed as shown by the increased Cal-520 AM fold change. F/F0 was used to investigate the calcium mobilization inside the aspirated RBC. From the above curves, the observed trend demonstrated that the Cal-520 AM signal increased proportionally with the increase of the applied negative aspiration pressure. Scale bar = 5 &#181;m. Abbreviation: RBCs = red blood cell. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66265/66265fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplemental Table S1: Checkpoints for critical steps in the fMPA preparation protocols. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66265/66265_Supplementary File - Table S1 - JoVE.xlsx" target="_blank">Please click here to download this File.</a>
Supplemental File 1: A guided process to adjust and perform segmentation. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66265/66265_Supp File 1.zip" target="_blank">Please click here to download this File.</a>

Watch this Scientific Journal Video about Advancing Live-Cell Mechanobiology Through Fluorescence Microaspiration at JoVE.com

Author Spotlight: Advancing Live-Cell Mechanobiology Through Fluorescence Microaspiration

The exploration of cellular behavior under mechanical stress is pivotal for advances in cellular mechanics and mechanobiology. We introduce the Fluorescence Micropipette Aspiration (fMPA) technique, a novel method combining controlled mechanical stimulation with comprehensive analysis of intracellular signaling in single cells. This technique investigates new in-depth studies of live-cell mechanobiology.

Fluorescence Micropipette Aspiration Assay to Investigate Red Blood Cell Mechanosensing

Micropipette aspiration assays have long been a cornerstone for the investigation of live-cell mechanics, offering insights into cellular responses to ...

fluorescence-micropipette-aspiration-assay-to-investigate-red-blood

Nanyang Technological University

Research

JoVE Journal

Biology

796 Views.  The University of Sydney. De reikwijdte van ons onderzoek is gericht op leefstijlmechanobiologie, in het bijzonder het gedrag van cellen waar mechanische belasting wordt toegepast. We onderzoeken de intracellulaire signalering in individuele cellen. Het voorgestelde FMPA-systeem maakt gebruik van fluorescentiebeeldvorming in combinatie met mechanische stimuli.Dat wil zeggen, de aspiratiedruk. Er zijn momenteel verdere ontwikkelingen die hebben geprobeerd om meer dan twee technieken te combineren met de microasp...

Video: Auteur in de schijnwerpers: het bevorderen van live-cell mechanobiologie door middel van fluorescentie-microaspiratie

Adhesion Frequency Assay for In Situ Kinetics Analysis of Cross-Junctional Molecular Interactions at the Cell-Cell Interface

The micropipette adhesion assay was developed in 1998 to measure two-dimensional (2D) receptor-ligand binding kinetics1. The assay uses a human red blood cell (RBC) as adhesion sensor and presenting cell for one of the interacting molecules. It employs micromanipulation to bring the RBC into contact with another cell that expresses the other interacting molecule with precisely controlled area and time to enable bond formation. The adhesion event is detected as RBC elongation upon pulling the two cells apart. By controlling the density of the ligands immobilized on the RBC surface, the probability of adhesion is kept in mid-range between 0 and 1. The adhesion probability is estimated from the frequency of adhesion events in a sequence of repeated contact cycles between the two cells for a given contact time. Varying the contact time generates a binding curve. Fitting a probabilistic model for receptor-ligand reaction kinetics1 to the binding curve returns the 2D affinity and off-rate.
The assay has been validated using interactions of Fc&gamma; receptors with IgG Fc1-6, selectins with glycoconjugate ligands6-9, integrins with ligands10-13, homotypical cadherin binding14, T cell receptor and coreceptor with peptide-major histocompatibility complexes15-19.
The method has been used to quantify regulations of 2D kinetics by biophysical factors, such as the membrane microtopology5, membrane anchor2, molecular orientation and length6, carrier stiffness9, curvature20, and impingement force20, as well as biochemical factors, such as modulators of the cytoskeleton and membrane microenvironment where the interacting molecules reside and the surface organization of these molecules15,17,19.
The method has also been used to study the concurrent binding of dual receptor-ligand species3,4, and trimolecular interactions19 using a modified model21.
The major advantage of the method is that it allows study of receptors in their native membrane environment. The results could be very different from those obtained using purified receptors17. It also allows study of the receptor-ligand interactions in a sub-second timescale with temporal resolution well beyond the typical biochemical methods.
To illustrate the micropipette adhesion frequency method, we show kinetics measurement of intercellular adhesion molecule 1 (ICAM-1) functionalized on RBCs binding to integrin &alpha;L&beta;2 on neutrophils with dimeric E-selectin in the solution to activate &alpha;L&beta;2.

Adhesion Frequency Assay for In Situ Kinetics Analysis of Cross-Junctional Molecular Interactions at the Cell-Cell Interface

An adhesion frequency assay for measuring receptor-ligand interaction kinetics when both molecules are anchored on the surfaces of the interacting cells is described. This mechanically-based assay is exemplified using a micropipette-pressurized human red blood cell as adhesion sensor and integrin &alpha;L&beta;2 and intercellular adhesion molecule-1 as interacting receptors and ligands.

The micropipette adhesion assay was developed in 1998 to measure two-dimensional (2D) receptor-ligand binding kinetics1. The assay uses a human red blood ...

Bioengineering

Micropipette Aspiration of Substrate-attached Cells to Estimate Cell Stiffness

Growing number of studies show that biomechanical properties of individual cells play major roles in multiple cellular functions, including cell proliferation, differentiation, migration and cell-cell interactions. The two key parameters of cellular biomechanics are cellular deformability or stiffness and the ability of the cells to contract and generate force. Here we describe a quick and simple method to estimate cell stiffness by measuring the degree of membrane deformation in response to negative pressure applied by a glass micropipette to the cell surface, a technique that is called Micropipette Aspiration or Microaspiration.
Microaspiration is performed by pulling a glass capillary to create a micropipette with a very small tip (2-50 &mu;m diameter depending on the size of a cell or a tissue sample), which is then connected to a pneumatic pressure transducer and brought to a close vicinity of a cell under a microscope. When the tip of the pipette touches a cell, a step of negative pressure is applied to the pipette by the pneumatic pressure transducer generating well-defined pressure on the cell membrane. In response to pressure, the membrane is aspirated into the pipette and progressive membrane deformation or "membrane projection" into the pipette is measured as a function of time. The basic principle of this experimental approach is that the degree of membrane deformation in response to a defined mechanical force is a function of membrane stiffness. The stiffer the membrane is, the slower the rate of membrane deformation and the shorter the steady-state aspiration length.The technique can be performed on isolated cells, both in suspension and substrate-attached, large organelles, and liposomes.
Analysis is performed by comparing maximal membrane deformations achieved under a given pressure for different cell populations or experimental conditions. A "stiffness coefficient" is estimated by plotting the aspirated length of membrane deformation as a function of the applied pressure. Furthermore, the data can be further analyzed to estimate the Young's modulus of the cells (E), the most common parameter to characterize stiffness of materials. It is important to note that plasma membranes of eukaryotic cells can be viewed as a bi-component system where membrane lipid bilayer is underlied by the sub-membrane cytoskeleton and that it is the cytoskeleton that constitutes the mechanical scaffold of the membrane and dominates the deformability of the cellular envelope. This approach, therefore, allows probing the biomechanical properties of the sub-membrane cytoskeleton.

Here we describe a quick and simple method to measure cell stiffness. The general principle of this approach is to measure membrane deformation in response to well-defined negative pressure applied through a micropipette to the cell surface. This method provides a powerful tool to study biomechanical properties of substrate-attached cells.

Growing number of studies show that biomechanical properties of individual cells play major roles in multiple cellular functions, including cell ...

Actin Co-Sedimentation Assay; for the Analysis of Protein Binding to F-Actin

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates tension and helps maintains cellular shape.  Although the actin cytoskeleton is a rigid structure, it is a dynamic structure that is constantly remodeling.  A number of proteins can bind to the actin cytoskeleton.  The binding of a particular protein to F-actin is often desired to support cell biological observations or to further understand dynamic processes due to remodeling of the actin cytoskeleton. The actin co-sedimentation assay is an in vitro assay routinely used to analyze the binding of specific proteins or protein domains with F-actin.  The basic principles of the assay involve an incubation of the protein of interest (full length or domain of) with F-actin, ultracentrifugation step to pellet F-actin and analysis of the protein co-sedimenting with F-actin.  Actin co-sedimentation assays can be designed accordingly to measure actin binding affinities and in competition assays.

Proteins bind to filamentous actin (F-actin) through distinct actin binding modules. In this video we demonstrate the procedure of actin co-sedimentation, which is an in vitro assay routinely used to analyze proteins or specific domains that bind F-actin.

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates ...

A Behavioral Assay to Measure Responsiveness of Zebrafish to Changes in Light Intensities 

The optokinetic reflex (OKR) is a basic visual reflex exhibited by most vertebrates and plays an important role in stabilizing the eye relative to the visual scene. However, the OKR requires that an animal detect moving stripes and it is possible that fish that fail to exhibit an OKR may not be completely blind. One zebrafish mutant, the no optokinetic response c (nrc) has no OKR under any light conditions tested and was reported to be completely blind. Previously, we have shown that OFF-ganglion cell activity can be recorded in these mutants. To determine whether mutant fish with no OKR such as the nrc mutant can detect simple light increments and decrements we developed the visual motor behavioral assay (VMR). In this assay, single zebrafish larvae are placed in each well of a 96-well plate allowing the simultaneous monitoring of larvae using an automated video-tracking system. The locomotor responses of each larva to 30 minutes light ON and 30 minutes light OFF were recorded and quantified. WT fish have a brief spike of motor activity upon lights ON, known as the startle response, followed by return to lower-than baseline activity, called a freeze. WT fish also sharply increase their locomotor activity immediately following lights OFF and only gradually (over several minutes) return to baseline locomotor activity. The nrc mutants respond similarly to light OFF as WT fish, but exhibit a slight reduction in their average activity as compared to WT fish. Motor activity in response to light ON in nrc mutants is delayed and sluggish. There is a slow rise time of the nrc mutant response to light ON as compared to WT light ON response. The results indicate that nrc fish are not completely blind. Because teleosts can detect light through non-retinal tissues, we confirmed that the immediate behavioral responses to light-intensity changes require intact eyes by using the chokh (chk) mutants, which completely lack eyes from the earliest stages of development. In our VMR assay, the chk mutants exhibit no startle response to either light ON or OFF, showing that the lateral eyes mediate this behavior. The VMR assay described here complements the well-established OKR assay, which does not test the ability of zebrafish larvae to respond to changes in light intensities. Additionally, the automation of the VMR assay lends itself to high-throughput screening for defects in light-intensity driven visual responses.

A Behavioral Assay to Measure Responsiveness of Zebrafish to Changes in Light Intensities

We developed the Visual-Motor Response to quantitate the motor output of larval zebrafish in response to light increments and decrements. We also examined zebrafish vision mutants, including the no optokinetic response (nrc) mutants, which were thought to be completely blind when tested by another vision assay, the optokinetic reflex.

The optokinetic reflex (OKR) is a basic visual reflex exhibited by most vertebrates and plays an important role in stabilizing the eye relative to the ...

Controlled Microfluidic Environment for Dynamic Investigation of Red Blood Cell Aggregation

Blood, as a non-Newtonian biofluid, represents the focus of numerous studies in the hemorheology field. Blood constituents include red blood cells, white blood cells and platelets that are suspended in blood plasma. Due to the abundance of the RBCs (40% to 45% of the blood volume), their behavior dictates the rheological behavior of blood especially in the microcirculation. At very low shear rates, RBCs are seen to assemble and form entities called aggregates, which causes the non-Newtonian behavior of blood. It is important to understand the conditions of the aggregates formation to comprehend the blood rheology in microcirculation. The protocol described here details the experimental procedure to determine quantitatively the RBC aggregates in microcirculation under constant shear rate, based on image processing. For this purpose, RBC-suspensions are tested and analyzed in 120 x 60 &#181;m poly-dimethyl-siloxane (PDMS) microchannels. The RBC-suspensions are entrained using a second fluid in order to obtain a linear velocity profile within the blood layer and thus achieve a wide range of constant shear rates. The shear rate is determined using a micro Particle Image Velocimetry (&#181;PIV) system, while RBC aggregates are visualized using a high speed camera. The videos captured of the RBC aggregates are analyzed using image processing techniques in order to determine the aggregate sizes based on the images intensities.

The protocol described details an experimental procedure to quantify Red Blood Cell (RBC) aggregates under a controlled and constant shear rate, based on image processing techniques. The goal of this protocol is to relate RBC aggregate sizes to the corresponding shear rate in a controlled microfluidic environment.

Blood, as a non-Newtonian biofluid, represents the focus of numerous studies in the hemorheology field. Blood constituents include red blood cells, white ...

Fluorescence Biomembrane Force Probe: Concurrent Quantitation of Receptor-ligand Kinetics and Binding-induced Intracellular Signaling on a Single Cell

Membrane receptor-ligand interactions mediate many cellular functions. Binding kinetics and downstream signaling triggered by these molecular interactions are likely affected by the mechanical environment in which binding and signaling take place. A recent study demonstrated that mechanical force can regulate antigen recognition by and triggering of the T-cell receptor (TCR). This was made possible by a new technology we developed and termed fluorescence biomembrane force probe (fBFP), which combines single-molecule force spectroscopy with fluorescence microscopy. Using an ultra-soft human red blood cell as the sensitive force sensor, a high-speed camera and real-time imaging tracking techniques, the fBFP is of ~1 pN (10-12 N), ~3 nm and ~0.5 msec in force, spatial and temporal resolution. With the fBFP, one can precisely measure single receptor-ligand binding kinetics under force regulation and simultaneously image binding-triggered intracellular calcium signaling on a single live cell. This new technology can be used to study other membrane receptor-ligand interaction and signaling in other cells under mechanical regulation.

We describe a technique for concurrently measuring force-regulated single receptor-ligand binding kinetics and real-time imaging of calcium signaling in a single T lymphocyte.

Membrane receptor-ligand interactions mediate many cellular functions. Binding kinetics and downstream signaling triggered by these molecular interactions ...

Mechano-Node-Pore Sensing: A Rapid, Label-Free Platform for Multi-Parameter Single-Cell Viscoelastic Measurements

Cellular mechanical properties are involved in a wide variety of biological processes and diseases, ranging from stem cell differentiation to cancer metastasis. Conventional methods for measuring these properties, such as atomic force microscopy (AFM) and micropipette aspiration (MA), capture rich information, reflecting a cell's full viscoelastic response; however, these methods are limited by very low throughput. High-throughput approaches, such as real-time deformability cytometry (RT-DC), can only measure limited mechanical information, as they are often restricted to single-parameter readouts that only reflect a cell's elastic properties. In contrast to these methods, mechano-node-pore sensing (mechano-NPS) is a flexible, label-free microfluidic platform that bridges the gap in achieving multi-parameter viscoelastic measurements of a cell with moderate throughput. A direct current (DC) measurement is used to monitor cells as they transit a microfluidic channel, tracking their size and velocity before, during, and after they are forced through a narrow constriction. This information (i.e., size and velocity) is used to quantify each cell's transverse deformation, resistance to deformation, and recovery from deformation. In general, this electronics-based microfluidic platform provides multiple viscoelastic cell properties, and thus a more complete picture of a cell's mechanical state. Because it requires minimal sample preparation, utilizes a straightforward electronic measurement (in contrast to a high-speed camera), and takes advantage of standard soft lithography fabrication, the implementation of this platform is simple, accessible, and adaptable to downstream analysis. This platform's flexibility, utility, and sensitivity have provided unique mechanical information on a diverse range of cells, with the potential for many more applications in basic science and clinical diagnostics.

Presented here is a method to mechanically phenotype single cells using an electronics-based microfluidic platform called mechano-node-pore sensing (mechano-NPS). This platform maintains moderate throughput of 1-10 cells/s while measuring both the elastic and viscous biophysical properties of cells.

Cellular mechanical properties are involved in a wide variety of biological processes and diseases, ranging from stem cell differentiation to cancer ...

Single Cell Durotaxis Assay for Assessing Mechanical Control of Cellular Movement and Related Signaling Events

Durotaxis is the process by which cells sense and respond to gradients of tension. In order to study this process in vitro, the stiffness of the substrate underlying a cell must be manipulated. While hydrogels with graded stiffness and long-term migration assays have proven useful in durotaxis studies, immediate, acute responses to local changes in substrate tension allow focused study of individual cell movements and subcellular signaling events. To repeatably test the ability of cells to sense and respond to the underlying substrate stiffness, a modified method for application of acute gradients of increased tension to individual cells cultured on deformable hydrogels is used which allows for real time manipulation of the strength and direction of stiffness gradients imparted upon cells in question. Additionally, by fine tuning the details and parameters of the assay, such as the shape and dimensions of the micropipette or the relative position, placement, and direction of the applied gradient, the assay can be optimized for the study of any mechanically sensitive cell type and system. These parameters can be altered to reliably change the applied stimulus and expand the functionality and versatility of the assay. This method allows examination of both long term durotactic movement as well as more immediate changes in cellular signaling and morphological dynamics in response to changing stiffness.

Mechanical forces are important for controlling cell migration. This protocol demonstrates the use of elastic hydrogels that can be deformed using a glass micropipette and a micromanipulator to stimulate cells with a local stiffness gradient to elicit changes in cell structure and migration.

Durotaxis is the process by which cells sense and respond to gradients of tension. In order to study this process in vitro, the stiffness of the substrate ...

Quantitative Analysis of Viscoelastic Properties of Red Blood Cells Using Optical Tweezers and Defocusing Microscopy

The viscoelastic properties of erythrocytes have been investigated by a range of techniques. However, the reported experimental data vary. This is not only attributed to the normal variability of cells, but also to the differences in methods and models of cell response. Here, an integrated protocol using optical tweezers and defocusing microscopy is employed to obtain the rheological features of red blood cells in the frequency range of 1 Hz to 35 Hz. While optical tweezers are utilized to measure the erythrocyte-complex elastic constant, defocusing microscopy is able to obtain the cell height profile, volume, and its form factor a parameter that allows conversion of complex elastic constant into complex shear modulus. Moreover, applying a soft glassy rheology model, the scaling exponent for both moduli can be obtained. The developed methodology allows to explore the mechanical behavior of red blood cells, characterizing their viscoelastic parameters, obtained under well-defined experimental conditions, for several physiological and pathological conditions.

Here, an integrated protocol based on optical tweezers and defocusing microscopy is described to measure the rheological properties of cells. This protocol has wide applicability in studying the viscoelastic properties of erythrocytes under variable physio-pathological conditions.

The viscoelastic properties of erythrocytes have been investigated by a range of techniques. However, the reported experimental data vary. This is not ...

Biomembrane Force Probe to Quantitate Receptor-Ligand Interactions

Source: Chen, Y., et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/52975">Fluorescence Biomembrane Force Probe: Concurrent Quantitation of Receptor-ligand Kinetics and Binding-induced Intracellular Signaling on a Single Cell.</a> J. Vis. Exp. (2015)
In this video, biomembrane force probes (BFPs) are used to measure the forces between receptors and ligands. This technique can detect interactions by monitoring the pressure needed to break the bonds between the pMHC conjugated on the probe beads and the TCRs on the target cells.

Source: Chen, Y., et al., Fluorescence Biomembrane Force Probe: Concurrent Quantitation of Receptor-ligand Kinetics and Binding-induced Intracellular ...

Fluorescence Micropipette Aspiration Assay to Investigate Red Blood Cell Mechanosensing

Samenvatting

Meer video's verkennen

Hoofdstukken in deze video