Name: measuring the ph, redox chemistries, and degradative capacity of macropinosomes using dual-fluorophore ratiometric microscopy
Uploaded: 2021-08-19T13:30:00.000+00:00
Duration: 7 min 31 s
Description: Watch this Scientific Journal Video about Measuring the pH, Redox Chemistries, and Degradative Capacity of Macropinosomes using Dual-Fluorophore Ratiometric Microscopy at JoVE.com

We thank the University of Calgary for its support. We would also like to thank Dr. Robin Yates for access to reagents, equipment, and useful discussions.

1. Preparation of cells
<ol>
	<li>Grow Raw264.7 cells at 37 &#176;C and 5% CO2 to 70% confluency in RPMI supplemented with 10% heat-inactivated serum.</li>
	<li>One day before the assay, seed Raw264.7 cells at a density of 5 x 104 cells per well in a 96-well plate. Ensure that each well contains 100 &#956;L of growth medium. Ensure that the 96-well plate has black sides and a glass-bottom for imaging. 
	NOTE: Here, 96-well plates were used for imaging. The 96-well plates allow for the use of smaller quantities of cells and reagents relative to larger imaging chambers. However, any chamber designed for imaging live cells can be used, and reagents can be scaled up accordingly.</li>
	<li>On the day of the assay, check whether the wells are at least 70% confluent.</li>
</ol>
2. Measuring macropinosome pH
<ol>
	<li>Prepare the cells as in section 1.</li>
	<li>Wash all the wells with 100 &#956;L of HBSS at 37 &#176;C.</li>
	<li>Fill each well with 100 &#956;L of HBSS containing 0.025 mg/mL of TMR-labeled 70 kDa dextran and 0.025 mg/mL of fluorescein-labeled 70 kDa dextran.</li>
	<li>Place the cells into an incubator set to 37 &#176;C for 15 min.</li>
	<li>Remove the cells from the incubator and wash the cells 6x with 100 &#956;L of HBSS.</li>
	<li>Fill each well with 100 &#956;L of HBSS at 37 &#176;C.</li>
	<li>Place the plate on the microscope with a heated stage and chamber.</li>
	<li>Adjust the excitation/emission parameters for each fluorophore as needed. 
	NOTE: Ideal conditions for image acquisition will vary with fluorophores used and individual microscope set-ups. Here, images were acquired on a Leica SP5 laser scanning confocal microscope. TMR was excited with a 543-laser line and emission was collected from 610 nm to 650 nm. Fluorescein was excited with a 488-laser line and emission was collected from 500 nm to 550 nm. A 63x oil immersion objective was used and a 10 &#956;m Z-volume was acquired at 0.5 &#956;m intervals.</li>
	<li>Acquire an image from each well, alternating between fluorophores in between each well.</li>
	<li>After the first acquisition, check whether all the wells remained in focus across the entire plate.</li>
	<li>Acquire images of each well at 1-15 min intervals for the desired length of time.</li>
</ol>
3. In situ calibration of macropinosome pH
<ol>
	<li>During the image acquisition, prepare 1 L of potassium-rich (K+-rich) solution containing 140 mM KCl, 1 mM MgCl2, 1 mM CaCl2, and 5 mM glucose. Supplement one 400 mL volume of the K+-rich solution with 25 mM HEPES (from a 1 M, pH 7.2 stock solution) and a separate 400 mL volume with 25 mM MES (from a 0.5 M, pH 6.0 stock solution).</li>
	<li>Adjust a 50 mL aliquot of the K+-rich solution buffered with 25mM HEPES to pH 7.5 using 10 M HCl or 10 M KOH as needed.</li>
	<li>Adjust three separate 50 mL aliquots of the K+-rich solution buffered with 25mM MES to pH 6.5, pH 5.5, and pH 5.0 using 10 M HCl or 10 M KOH as needed. 
	NOTE: An acetate-acetic acid buffer may be preferable for lower pH solutions.</li>
	<li>After the completion of image acquisition, remove the HBSS from the 96-well plate containing the cells and replace it with the K+-rich solution that is at pH 7.5.</li>
	<li>Add nigericin to a final concentration of 10 &#956;g/mL. 
	NOTE: Nigericin is an ionophore that exchanges K+ for H+. By setting the K+ concentration of the K+-rich solution to a value that approximates the K+ concentration of the cytosol, it can be ensured that nigericin will clamp the pH of the macropinosome to that of the cytosol, which in turn reflects the pH of the calibration buffer.</li>
	<li>Place the plate back on the microscope and acquire images of each well using the same acquisition settings as above.</li>
	<li>Repeat steps 3.5 and 3.6 for each calibration buffer.</li>
</ol>
4. Data analysis for macropinosome pH
<ol>
	<li>Using FIJI software, subtract the background from both the TMR and the fluorescein channel.</li>
	<li>Generate a mask on the TMR channel. To generate a mask, convert the image to a binary image by selecting Adjust &#62; Threshold &#62; Apply. Next, highlight the binary image and select Edit &#62; Selection &#62; Create Mask.</li>
	<li>Apply it to both the TMR and fluorescein images. To do this, highlight the mask and select Edit &#62; Selection &#62; Create Selection. Click on the TMR image and press Shift + E to apply the mask to the TMR channel. Similarly, click on the fluorescein image and press Shift + E to apply the mask to the OB channel.</li>
	<li>Record the TMR and fluorescein intensity for each macropinosome within the masks.</li>
	<li>Repeat steps 4.1-4.3 for each time point from the time course.</li>
	<li>Repeat steps 4.1-4.3 for the images captured in the in situ calibration.</li>
	<li>Divide the fluorescein intensity by the TMR intensity to generate the fluorescein:TMR ratio.</li>
	<li>Plot the pH against the fluorescein:TMR ratios for the calibration images.</li>
	<li>Fit a curve to the calibration data.</li>
	<li>Interpolate the data for each time point from the time course to get absolute pH values.</li>
</ol>
5. Measuring oxidative events within macropinosomes
<ol>
	<li>Prepare the cells as in section 1.</li>
	<li>One day before the assay, prepare the H2DCFDA succinimidyl ester labeled 70 kDa dextran by resuspending 10 mg of 70 kDa dextran-amino in 1 mL of 0.1 M sodium bicarbonate solution (pH 8.3). Add 1 mg of the H2DCFDA succinimidyl ester to the dextran-amino solution and incubate for 1 h. Dialyze the H2DCFDA-labeled 70 kDa dextran against PBS. Do not store for more than 1 day as H2DCFDA-labeled 70 kDa dextran will oxidize upon storage.</li>
	<li>On the day of the assay, wash all the wells with 100 &#956;L of HBSS at 37 &#176;C.</li>
	<li>Fill each well with 100 &#956;L of HBSS containing 0.025 mg/mL of TMR-labeled 70 kDa dextran and 0.025 mg/mL of H2DCFDA-labeled 70 kDa dextran.</li>
	<li>Place cells into an incubator set to 37 &#176;C for 15 min.</li>
	<li>Remove cells from the incubator and wash the cells 6x with 100 &#956;L of HBSS.</li>
	<li>Fill each well with 100 &#956;L of HBSS at 37 &#176;C.</li>
	<li>Place the plate on the microscope with a heated stage and chamber and adjust the excitation/emission parameters for each fluorophore as needed. 
	NOTE: Ideal conditions for image acquisition will vary with fluorophores used and individual microscope set-ups. Here images were acquired on a Leica SP5 laser scanning confocal microscope. TMR was excited with a 543-laser line, and emission was collected from 610 nm to 650 nm. H2DCFDA was excited with a 488-laser line and emission was collected from 500 nm to 550 nm. A 63x oil immersion objective was used, and a 10 &#956;m Z-volume was acquired at 0.5 &#956;m intervals.</li>
	<li>Acquire an image from each well, alternating between fluorophores in between each well.</li>
	<li>After the first acquisition, check whether all the wells remain in focus across the entire plate.</li>
	<li>Acquire images of each well at 1-15 min intervals for the desired length of time.</li>
</ol>
6. Data analysis for oxidative events within macropinosomes
<ol>
	<li>Using FIJI software, subtract the background from both the TMR and H2DCFDA channels.</li>
	<li>Generate a mask on the TMR channel. To generate a mask, convert the image to a binary image by selecting Adjust &#62; Threshold &#62; Apply. Next, highlight the binary image and select Edit &#62; Selection &#62; Create Mask.</li>
	<li>Apply the mask to both the TMR and H2DCFDA images. To do this, highlight the mask and select Edit &#62; Selection &#62; Create Selection. Click on the TMR image and press Shift + E to apply the mask to the TMR channel. Similarly, click on the H2DCFDA image and press Shift + E to apply the mask to the H2DCFDA channel.</li>
	<li>Record the TMR and H2DCFDA intensity for each macropinosome within the masks.</li>
	<li>Repeat steps 4.1-4.3 for each time point from the time course.</li>
	<li>Divide the H2DCFDA intensity by the TMR intensity to generate an H2DCFDA: TMR ratio.</li>
	<li>Plot the H2DCFDA: TMR ratio against time.</li>
</ol>
7. Measuring protein digestion within macropinosomes
<ol>
	<li>Prepare the cells as in section 1.</li>
	<li>On the day of the assay, wash all the wells with 100 &#956;L HBSS at 37 &#176;C.</li>
	<li>Dissolve BODIPY-labeled ovalbumin to a concentration of 4 mg/mL in HBSS.</li>
	<li>Fill each well with 100 &#956;L of HBSS containing 0.025 mg/mL of TMR-labeled 70 kDa dextran and 0.2 mg/mL of BODIPY-labeled ovalbumin.</li>
	<li>Place the cells into an incubator set to 37 &#176;C for 15 min.</li>
	<li>Remove cells from the incubator and wash the cells 6x with 100 &#956;L of HBSS.</li>
	<li>Fill each well with 100 &#956;L of HBSS at 37 &#176;C.</li>
	<li>Place the plate on the microscope with a heated stage and chamber and adjust the excitation/emission parameters for each fluorophore as needed. 
	NOTE: Ideal conditions for image acquisition will vary with fluorophores used and individual microscope set-ups. Here, images were acquired on a Leica SP5 laser scanning confocal microscope. TMR was excited with a 543-laser line, and emission was collected from 610 nm to 650 nm. BODIPY was excited with a 488-laser line, and emission was collected from 500 nm to 550 nm. A 63x oil immersion objective was used and a 10 &#956;m Z-volume was acquired at 0.5 &#956;m intervals.</li>
	<li>Acquire an image from each well, alternating between fluorophores in between each well.</li>
	<li>After the first acquisition, check whether all the wells remain in focus across the entire plate.</li>
	<li>Acquire images of each well at 1-15 min intervals for the desired length of time.</li>
</ol>
8. Data analysis for protein digestion within macropinosomes
<ol>
	<li>Using FIJI software, subtract the background from both the TMR and BODIPY channels.</li>
	<li>Generate a mask on the TMR channel and apply it to both the TMR and BODIPY images as in steps 6.2 and 6.3 above (Figure 3B).</li>
	<li>Record the TMR and BODIPY intensity for each macropinosome within the masks.</li>
	<li>Repeat steps 4.1-4.3 for each time point from the time course.</li>
	<li>Divide the BODIPY intensity by the TMR intensity to generate the BODIPY:TMR ratio.</li>
	<li>Plot the BODIPY:TMR ratio against time.</li>
</ol>

<ol>
	<li>Canton, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macropinocytosis:+New+insights+into+its+underappreciated+role+in+innate+immune+cell+surveillance.">Macropinocytosis: New insights into its underappreciated role in innate immune cell surveillance.</a> Frontiers in Immunology. 9, (2018).</li><li>Marques, P. E., Grinstein, S., Freeman, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SnapShot:+Macropinocytosis.">SnapShot: Macropinocytosis.</a> Cell. 169, 766 (2017).</li><li>Williams, T., Kay, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+measurement+of+Dictyostelium+discoideum+macropinocytosis+by+flow+cytometry.">High-throughput measurement of Dictyostelium discoideum macropinocytosis by flow cytometry.</a> Journal of Visualized Experiments: JoVE. (139), e58434 (2018).</li><li>Ganot, P., 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=Ubiquitous+macropinocytosis+in+anthozoans.">Ubiquitous macropinocytosis in anthozoans.</a> eLife. 50022, (2020).</li><li>Charpentier, J. C., 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=Macropinocytosis+drives+T+cell+growth+by+sustaining+the+activation+of+mTORC1.">Macropinocytosis drives T cell growth by sustaining the activation of mTORC1.</a> Nature Communications. 11, 180 (2020).</li><li>Bohdanowicz, 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=Phosphatidic+acid+is+required+for+the+constitutive+ruffling+and+macropinocytosis+of+phagocytes.">Phosphatidic acid is required for the constitutive ruffling and macropinocytosis of phagocytes.</a> Molecular Biology of the Cell. 24, 1700-1712 (2013).</li><li>Commisso, C., Flinn, R. J., Bar-Sagi, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determining+the+macropinocytic+index+of+cells+through+a+quantitative+image-based+assay.">Determining the macropinocytic index of cells through a quantitative image-based assay.</a> Nature Protocols. 9, 182-192 (2014).</li><li>Yoshida, S., Pacitto, R., Sesi, C., Kotula, L., Swanson, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dorsal+ruffles+enhance+activation+of+Akt+by+growth+factors.">Dorsal ruffles enhance activation of Akt by growth factors.</a> Journal of Cell Science. 131, (2018).</li><li>Commisso, C., 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=Macropinocytosis+of+protein+is+an+amino+acid+supply+route+in+Ras-transformed+cells.">Macropinocytosis of protein is an amino acid supply route in Ras-transformed cells.</a> Nature. 497, 633-637 (2013).</li><li>Commisso, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pervasiveness+of+macropinocytosis+in+oncological+malignancies.">The pervasiveness of macropinocytosis in oncological malignancies.</a> Philosophical Transactions of the Royal Society of London B Biological Sciences. 374, 20180153 (2019).</li><li>Zhang, 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=Macropinocytosis+in+cancer-associated+fibroblasts+is+dependent+on+CaMKK2/ARHGEF2+signaling+and+functions+to+support+tumor+and+stromal+cell+fitness.">Macropinocytosis in cancer-associated fibroblasts is dependent on CaMKK2/ARHGEF2 signaling and functions to support tumor and stromal cell fitness.</a> Cancer Discovery. , (2021).</li><li>Lee, S. -. W., 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=EGFR-Pak+signaling+selectively+regulates+glutamine+deprivation-induced+macropinocytosis.">EGFR-Pak signaling selectively regulates glutamine deprivation-induced macropinocytosis.</a> Developmental Cell. 50, 381-392 (2019).</li><li>Michalopoulou, E., 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=Macropinocytosis+renders+a+subset+of+pancreatic+tumor+cells+resistant+to+mTOR+inhibition.">Macropinocytosis renders a subset of pancreatic tumor cells resistant to mTOR inhibition.</a> Cell Reports. 30, 2729-2742 (2020).</li><li>Mercer, J., Helenius, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Virus+entry+by+macropinocytosis.">Virus entry by macropinocytosis.</a> Nature Cell Biology. 11, 510-520 (2009).</li><li>West, M. A., 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=Enhanced+dendritic+cell+antigen+capture+via+toll-like+receptor-induced+actin+remodeling.">Enhanced dendritic cell antigen capture via toll-like receptor-induced actin remodeling.</a> Science. 305, 1153-1157 (2004).</li><li>Canton, J., 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=Calcium-sensing+receptors+signal+constitutive+macropinocytosis+and+facilitate+the+uptake+of+NOD2+ligands+in+macrophages.">Calcium-sensing receptors signal constitutive macropinocytosis and facilitate the uptake of NOD2 ligands in macrophages.</a> Nature Communications. 7, 11284 (2016).</li><li>Sallusto, F., Cella, M., Danieli, C., Lanzavecchia, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic+cells+use+macropinocytosis+and+the+mannose+receptor+to+concentrate+macromolecules+in+the+major+histocomaptibility+complex+class+II+compartment:+downregulation+by+cytokines+and+bacterial+products.">Dendritic cells use macropinocytosis and the mannose receptor to concentrate macromolecules in the major histocomaptibility complex class II compartment: downregulation by cytokines and bacterial products.</a> Journal of Experimental Medicine. 182, 389-400 (1995).</li><li>Bhosle, V. K., 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=SLIT2/ROBO1-signaling+inhibits+macropinocytosis+by+opposing+cortical+cytoskeletal+remodeling.">SLIT2/ROBO1-signaling inhibits macropinocytosis by opposing cortical cytoskeletal remodeling.</a> Nature Communications. 11, 4112 (2020).</li><li>Liu, Z., Roche, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macropinocytosis+in+phagocytes:+regulation+of+MHC+class-II-restricted+antigen+presentation+in+dendritic+cells.">Macropinocytosis in phagocytes: regulation of MHC class-II-restricted antigen presentation in dendritic cells.</a> Frontiers in Physiology. 6, 1 (2015).</li><li>Desai, A. S., Hunter, M. R., Kapustin, A. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+macropinocytosis+for+intracellular+delivery+of+therapeutic+nucleic+acids+to+tumour+cells.">Using macropinocytosis for intracellular delivery of therapeutic nucleic acids to tumour cells.</a> Philosophical Transactions of the Royal Society of London B. Biological Sciences. 374, 20180156 (2019).</li><li>Freeman, S. A., 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=Lipid-gated+monovalent+ion+fluxes+regulate+endocytic+traffic+and+support+immune+surveillance.">Lipid-gated monovalent ion fluxes regulate endocytic traffic and support immune surveillance.</a> Science. 367, 301-305 (2020).</li><li>Canton, J., Grinstein, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+phagosomal+pH+by+fluorescence+microscopy.">Measuring phagosomal pH by fluorescence microscopy.</a> Methods in Molecular Biology. 1519, 185-199 (2017).</li><li>Cheung, S., Greene, C., Yates, 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=Simultaneous+analysis+of+multiple+lumenal+parameters+of+individual+phagosomes+using+high-content+imaging.">Simultaneous analysis of multiple lumenal parameters of individual phagosomes using high-content imaging.</a> Methods Molecular Biology. 1519, 227-239 (2017).</li><li>Li, 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=The+effect+of+the+size+of+fluorescent+dextran+on+its+endocytic+pathway.">The effect of the size of fluorescent dextran on its endocytic pathway.</a> Cell Biology International. 39, 531-539 (2015).</li><li>Canton, J., Grinstein, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+lysosomal+pH+by+fluorescence+microscopy.">Measuring lysosomal pH by fluorescence microscopy.</a> Methods in Cell Biology. 126, 85-99 (2015).</li><li>Armstrong, J. K., Wenby, R. B., Meiselman, H. J., Fisher, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+hydrodynamic+radii+of+macromolecules+and+their+effect+on+red+blood+cell+aggregation.">The hydrodynamic radii of macromolecules and their effect on red blood cell aggregation.</a> Biophysical Journal. 87, 4259-4270 (2004).</li><li>Blum, J. S., Wearsch, P. A., Cresswell, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathways+of+antigen+processing.">Pathways of antigen processing.</a> Annual Reviews of Immunology. 31, 443-473 (2013).</li><li>Canton, J., Khezri, R., Glogauer, M., Grinstein, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contrasting+phagosome+pH+regulation+and+maturation+in+human+M1+and+M2+macrophages.">Contrasting phagosome pH regulation and maturation in human M1 and M2 macrophages.</a> Molecular Biology of the Cell. 25, 3330-3341 (2014).</li><li>Mantegazza, A. R., 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=NADPH+oxidase+controls+phagosomal+pH+and+antigen+cross-presentation+in+human+dendritic+cells.">NADPH oxidase controls phagosomal pH and antigen cross-presentation in human dendritic cells.</a> Blood. 112, 4712-4722 (2008).</li><li>Canton, J., 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=The+receptor+DNGR-1+signals+for+phagosomal+rupture+to+promote+cross-presentation+of+dead-cell-associated+antigens.">The receptor DNGR-1 signals for phagosomal rupture to promote cross-presentation of dead-cell-associated antigens.</a> Nature Immunology. 22, 140-153 (2021).</li><li>Mishra, R., Bhowmick, N. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+macropinocytosis+in+prostate+fibroblasts.">Visualization of macropinocytosis in prostate fibroblasts.</a> Bio-Protocol. 9, (2019).</li><li>Galenkamp, K. M. O., Alas, B., Commisso, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitation+of+macropinocytosis+in+cancer+cells.">Quantitation of macropinocytosis in cancer cells.</a> Methods in Molecular Biology. 1928, 113-123 (2019).</li><li>Lee, S. -. W., Alas, B., Commisso, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+and+quantification+of+macropinosomes+in+pancreatic+tumors.">Detection and quantification of macropinosomes in pancreatic tumors.</a> Methods in Molecular Biology. 1882, 171-181 (2019).</li><li>Swanson, J. A., King, J. 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+breadth+of+macropinocytosis+research.">The breadth of macropinocytosis research.</a> Philosophical Transactions of the Royal Society of London B. Biological Sciences. 374, 20180146 (2019).</li></ol>

The authors have no conflicts of interest to disclose.

Although there are a number of protocols for both low and high-throughput measurements of macropinocytic uptake in macrophages, fibroblasts, and even Dictyostelium spp.3,7,31,32,33, very few attempts have been made to measure the luminal biochemistry of these dynamic compartments. This is likely due to a paucity of probes that can be efficiently targeted to the macropinosomal compartment while avoiding other endocytic compartments. Described here are the techniques to target fluorescent probes for the dynamic measurement of pH, oxidative events, and protein digestion within macropinosomes.
Ratiometric fluorescence microscopy has been the primary technique by which endosomal pH has been measured for decades. Fluorescein has been widely used for measuring endosomal pH and continues to be the fluorophore of choice as its pKa allows for accurate measurements with the typical range of endosomal pH values (~pH 7.2-4.5). Here, the study employed fluorescein conjugated to 70 kDa dextran for macropinosome-specific pH measurements. A limitation of this approach is the 15 min dextran loading period during which macropinosomal pH cannot be recorded. This period is required to load sufficient fluorophore-labeled dextran into the macropinosomal compartment but effectively precludes early macropinosome pH measurements. This does not, however, preclude determinations of various luminal parameters such as the buffering capacity, relative proton leak, and V-ATPase pumping power, all of which are derived from macropinosomal pH measurements. As shown in Figure 1, a number of other pH-sensitive fluorophores may also be used to measure pH, including pHrodo (pKa = 6.8) and CypHer5e (pKa = 7.3). However, as they have a significantly higher pKa than fluorescein, they are not optimal for measuring endosomal pH at more acidic pH values.
As macropinosomes are highly dynamic organelles and undergo significant shrinkage shortly after forming16,21, dual-fluorophore ratiometric fluorescence is required. Changes in fluorescence due to changes in the volume of macropinosomes are corrected for using a reference fluorophore in all of the assays presented.
Oxidative events in cells have been measured using a variety of redox-sensitive probes, including nitroblue tetrazolium (NBT), luminol, and H2DCFDA. Luminol-based approaches are particularly useful for measuring ROS production in a population of cells but are difficult to adapt to organelle-specific measurements. NBT forms an easily visualized insoluble deposit called formazan upon oxidation but obscures fluorescent signals and is both nonlinear and difficult to localize. H2DCFDA liberates a linear fluorescent signal upon oxidation and can be readily conjugated to tracers such as dextran for organelle-specific measurements. By attaching H2DCFDA to 70 kDa dextran, oxidative events within individual macropinosomes (Figure 3B) can be visualized. There is, however, a caveat to this approach. As H2DCFDA is a modified form of fluorescein, the signal liberated is pH sensitive. Although the gain in fluorescence upon oxidation is large enough to be detected, it is undoubtedly masked by the acidic lumen of the macropinosome. A pH-insensitive variant of H2DCFDA has been previously commercially available and is preferable; however, at the time of preparing this manuscript, this product was no longer commercially available. ROS is emerging as an important signaling molecule and has been implicated in membrane remodeling, organellar trafficking, and more recently in the processing of the material by antigen-presenting cells for presentation to T cells28,30. The method presented here can be used to study the contribution of macropinosomal ROS production to these various physiological processes.
Protein digestion in macropinosomes is of interest in the study of antigen presentation by immune cells as well as the study of nutrient acquisition by cancer cells. For example, endosomal protein digestion by dendritic cells is believed to limit the pool of peptides available for presentation to T cells on major histocompatibility molecules. As macropinosomes are a major route of antigen acquisition, measurements of macropinosomal protein digestion may be used to probe the molecular machinery responsible for fine-tuning antigen presentation. A number of commercially available tools are used for measuring protein digestion in endosomal compartments. The BODIPY-labeled BSA and ovalbumin probes, which become strongly fluorescent upon digestion, have proven particularly popular. They are easily coupled to endocytic tracers and can be targeted to specific endosomal compartments, such as phagosomes. The probes, however, are not readily coupled to dextran and so require a co-localization approach. This approach results in the loading of all endosomal compartments, including macropinosomes, with BODIPY-labeled ovalbumin, and a mask is used to measure the macropinosome-specific degradation. This approach requires better resolution and is not easily scaled up for larger, more high throughput analyses. Techniques to covalently couple protein degradation tools to 70 kDa dextran will likely prove useful and are currently being developed in our laboratory.
As the field of macropinocytosis continues to grow34, tools for dynamically measuring the luminal biochemistry of macropinosomes, including pH, redox chemistries, protein digestion, ion concentrations, and lipid chemistries will provide insight into the unique biology of these rapidly evolving organelles.

Macropinocytosis refers to the uptake of large quantities of extracellular fluid into membrane-bound cytoplasmic organelles called macropinosomes1,2. It is a highly conserved process performed by free-living unicellular organisms, such as the amoeba Dictyostelium spp.3, as well as anthozoans4 and metazoans2. In most cells, macropinocytosis is an induced event. The ligation of cell-surface receptors induces the protrusion of actin-driven plasma membrane extensions referred to as ruffles. A fraction of those ruffles, by some poorly understood mechanism, seal at their distal tips to form macropinosomes (although beyond the scope of this methods paper, for detailed reviews on the mechanics of macropinocytosis, please refer to references1,2,5,6,7). The extracellular stimulus inducing macropinocytosis is most often a soluble growth factor5,8. Accordingly, the macropinocytic event allows for the ingestion of a bolus of extracellular material from which the cell can derive useful metabolites to facilitate growth. Unfortunately, this pathway for nutrient delivery can also drive pathology. Certain cancer cells harbor mutations that result in continuous or constitutive macropinocytosis. The continuous delivery of nutrients facilitates the uncontrolled proliferation of cancer cells and has been linked to particularly aggressive tumors9,10,11,12,13. Similarly, viruses can induce macropinocytosis to gain access to host cells, thereby driving viral pathology14.
Macropinocytosis also functions in the maintenance of immunity to pathogens. Certain innate immune cells such as macrophages and dendritic cells engage in the constitutive and aggressive sampling of extracellular fluid via macropinocytosis6,15,16. This mode of macropinocytosis is incredibly active, and a single dendritic cell can engorge itself with a volume of extracellular fluid equivalent to its own weight every hour17. Despite this constitutive sampling, macrophages and dendritic cells do not replicate uncontrollably as do tumor cells, instead, they seem to process the extracellular material in such a way that information can be extracted to inform upon the presence, or indeed absence, of potential threats. Information is extracted as i) pathogen-associated molecular patterns that can be read by intracellular pathogen recognition receptors and ii) short stretches of amino acids that can be loaded onto major histocompatibility molecules for screening by cells of the adaptive immune system16,18,19. Whether pathogens subvert this pathway for information processing by immune cells is at present unclear.
Despite these well-defined and critical roles for macropinocytosis in both the maintenance of immunity and homeostasis and in contrast to other more commonly studied modes of endocytosis, little of the inner (luminal) workings of macropinosomes is known. Developing standardized protocols and tools to study the luminal biochemistry of macropinosomes will not only help us to understand their unique biology better but will provide insight that can be leveraged for novel therapeutic strategies, including drug delivery20. This method manuscript will focus on recently developed tools to dissect, at the single organelle level, various aspects of the luminal biochemistry of macropinosomes.
Fluorophores can be used to measure specific biochemistries of organelles if i) they partition preferentially into the compartment of interest and/or ii) they undergo spectral changes in response to the parameter of interest. For example, in the case of pH, fluorescent weak bases, such as acridine orange, cresyl violet, and the LysoTracker dyes accumulate preferentially in acidic organelles. Therefore, their relative intensity is a rough indication that the labeled organelle is acidic. Other pH-responsive fluorophores, such as fluorescein, pHrodo, and cypHer5e, undergo spectral changes upon binding to protons (Figure 1A-C). Changes to the fluorescence emission of pH-sensitive fluorophores can therefore provide a useful approximation of pH. The use of single fluorophores, however, presents a number of disadvantages. For example, changes to the focal plane, photobleaching, and changes to the volume of individual organelles, a common occurrence in macropinosomes21, can induce changes to the fluorescence intensity of single fluorophores, and this cannot be easily corrected for22. Single-wavelength assessments, although useful for visualizing acidic compartments, are therefore purely qualitative.
A more quantitative approach is to target the parameter-sensitive fluorophore along with a reference fluorophore to the organelle of interest. The reference fluorophore is ideally insensitive to biochemical changes within the organelle (Figure 1D-F) and can therefore be used to correct for changes in the focal plane, organellar volume, and, to some extent, photobleaching23. Using this approach, referred to as dual-fluorophore ratiometric fluorescence, correction can be achieved by generating a ratio of the fluorescence emission of the parameter-sensitive fluorophore to the reference fluorophore.
Here, the protocol will be utilizing the principle of dual-fluorophore ratiometric imaging to measure pH, oxidative events, and protein degradation within macropinosomes. In each case, a fluorophore will be selected that is sensitive to the parameter of interest and a reference fluorophore. In order to target the fluorophores specifically to macropinosomes, they will be covalently coupled to 70 kDa dextran, which is preferentially incorporated into macropinosomes24. All assays will be performed in Raw264.7 cells but can be adapted to other cell types. Where possible, the fluorescence ratios will be calibrated against a reference curve to gain absolute values. Importantly, all measurements will be performed in live cells for dynamic and quantitative assessment of the luminal environment of macropinosomes.
When selecting pH-sensitive fluorophores, a number of considerations must be weighed. The first is the pKa of the fluorophore, which indicates the range of pH values at which the probe will be most sensitive. If it is assumed that shortly after formation, the pH of the macropinosome will be close to that of the extracellular medium (~pH 7.2) and that it will progressively acidify through interactions with late endosomes and lysosomes (~pH 5.0), then a probe with a pKa that is sensitive within that range (Figure 2C) should be selected. The fluorophore fluorescein, which has a pKa of 6.4, is optimally sensitive within that range. It has been used extensively to measure other similar organelles, such as phagosomes, and will be the fluorophore of choice in this manuscript22,25. As a reference fluorophore, tetramethylrhodamine will be used, which is insensitive to pH (Figure 1E). Other fluorophores, such as pHrodo and cypHer5e may be substituted for fluorescein where the spectral properties of fluorescein do match other experimental variables. Some suggested reference fluorophores for pHrodo and cypHer5e are shown in Figure 1.
A second consideration is the method by which the two fluorophores will be targeted specifically to macropinosomes. Dextran of the size 70 kDa, which has a hydrodynamic radius of roughly 7 nm, does not stick non-specifically to cells and is incorporated into macropinosomes, but not clathrin-coated pits or caveolae, and therefore marks macropinosomes (Figure 2A and Figure 3A,B)16,24,26. In this protocol, fluorescein-labeled 70 kDa dextran and tetramethylrhodamine (TMR)-labeled 70 kDa dextran will be used as the pH-sensitive and reference probes, respectively.
In innate immune cells, macropinocytosis and phagocytosis represent the two major routes for the internalization of exogenous material for processing and subsequent presentation to cells of the adaptive immune response27. The careful and coordinated control of the redox chemistry of the lumen of phagosomes and macropinosomes is critical to the context-specific processing of exogenous material. Perhaps the most well-studied regulator of oxidative events in phagosomes is the NADPH oxidase, a large multi-subunit complex that produces large quantities of reactive oxygen species (ROS) within the lumen of phagosomes28. Indeed, its activity is central to appropriate antigen processing within phagosomes29,30. Yet, the activity of the NADPH oxidase on macropinosomal membranes has not been explored.
In this protocol, the H2DCFDA succinimidyl ester is used for measuring oxidative events within the macropinosome. This is a modified form of fluorescein (2&#39;,7&#39;-dichlorodihydrofluorescein diacetate), which is minimally fluorescent in its reduced form. Upon oxidation, its fluorescence emission increases significantly. It is, however, worth noting a significant caveat of H2DCFDA - as it is based on the fluorophore fluorescein, its fluorescence is also quenched in acidic compartments, and care must be taken to control for this variable when designing experiments28. Similar to the approach for measuring pH, the H2DCFDA succinimidyl ester will be covalently attached to 70 kDa dextran and TMR-labeled 70 kDa dextran will be used as the reference fluorophore (Figure 3A).
Fluorescent ovalbumin will be used to measure protein degradation within macropinosomes. The ovalbumin used here is densely labeled with a 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) FL dye that is self-quenched. Upon digestion, strongly fluorescent dye-labeled peptides are liberated. As ovalbumin cannot be easily conjugated to 70 kDa dextran, cells with TMR-labeled 70 kDa dextran and fluid-phase ovalbumin will be co-incubated. The TMR signal will be used to generate a macropinosome mask during post-imaging analysis, and the signal liberated from the digested ovalbumin will be measured within the mask (Figure 3B).

<table><tbody><tr><td>Black-walled 96 well plate</td><td>PerkinElmer</td><td>6005430</td><td></td></tr><tr><td>CypHer5e, NHS ester</td><td>Cytiva</td><td>PA15401</td><td></td></tr><tr><td>Dextran-amino 70 kDa</td><td>Invitrogen</td><td>D1862</td><td></td></tr><tr><td>DQ-ovalbumin</td><td>Invitrogen</td><td>D12053</td><td></td></tr><tr><td>FITC-dextran 70 kDa</td><td>Invitrogen</td><td>D1823</td><td></td></tr><tr><td>HBSS</td><td>Gibco</td><td>14287</td><td></td></tr><tr><td>Nigericin</td><td>Sigma Aldrich</td><td>N7143</td><td></td></tr><tr><td>OxyBurst Green-SE</td><td>Invitrogen</td><td>D2935</td><td></td></tr><tr><td>pHrodo Red, SE</td><td>Invitrogen</td><td>P36600</td><td></td></tr><tr><td>Raw264.7 cells</td><td>ATCC</td><td>TIB-71</td><td></td></tr><tr><td>RPMI medium</td><td>Gibco</td><td>11875093</td><td></td></tr><tr><td>SP5 Confocal Microscope</td><td>Leica</td><td>-</td><td></td></tr><tr><td>TRITC-dextran 70 kDa</td><td>Invitrogen</td><td>D1819</td><td></td></tr><tr><td>u-Dish</td><td>Ibidi</td><td>81156</td><td></td></tr></tbody></table>

measuring the ph, redox chemistries, and degradative capacity of macropinosomes using dual-fluorophore ratiometric microscopy

In recent years, the field of macropinocytosis has grown rapidly. Macropinocytosis has emerged as a central mechanism by which innate immune cells maintain organismal homeostasis and immunity. Simultaneously, and in contrast to its homeostatic role, it can also drive various pathologies, including cancer and viral infections. Unlike other modes of endocytosis, the tools developed for studying the maturation of macropinosomes remain underdeveloped. Here the protocol describes newly developed tools for studying the redox environment within the lumen of early and maturing macropinosomes. Methodologies for using ratiometric fluorescence microscopy in assessing the pH, production of reactive oxygen species, and the degradative capacity within the lumen of individual macropinosomes in live cells are described. Single organelle measurements offer the advantage of revealing spatiotemporal heterogeneity, which is often lost with population-based approaches. Emphasis is placed on the basic principles of dual fluorophore ratiometric microscopy, including probe selection, instrumentation, calibration, and single-cell versus population-based methods.

When measuring macropinosome pH, there is a period of time for which the dynamics of acidification cannot be measured. This period corresponds to the dextran loading phase (gray box in Figure 2C and Figure 3C,D) and will vary depending on the cell type used. The length of the loading phase will vary with (i) the macropinocytic activity of the cells and (ii) the sensitivity of the instrument used. It is recommended to adjust this period at the start of each experiment such that a signal:noise ratio of greater than 4:1 is achieved for both the fluorescein and the TMR channels. In untreated cells, the fluorescein emission should become progressively weaker as the macropinosomes acidify, and the TMR signal should remain either constant or become brighter. The TMR signal may become brighter if the macropinosomes shrink in size16,21. This will not affect the ratio as the fluorescein signal is subject to the same variations in organellar size22. The fluorescein:TMR ratio will become progressively smaller and will plateau within the first 15 min of acquisition (Figure 2C). This plateau corresponds to a pH of ~5, which roughly matches the pH of lysosomes25. At the end of each acquisition, an in situ calibration is performed. The fluorescein:TMR ratio should be largest with the calibration buffer at pH 7.5 and become progressively smaller as the calibration buffers become more acidic. This allows for the generation of a calibration curve (Figure 2B). The fluorescein:TMR ratios can then be interpolated for macropinosomal pH (Figure 2C).
Oxidative events within macropinosomes are also likely to vary with the cell type being used. It is recommended to load the cells with H2DCFDA-dextran, and to stimulate the NADPH oxidase using phorbol 12-myristate 13-acetate (PMA) as a positive control (Figure 3C). This will give a better idea of the dynamic range of the assay and will allow for adjustment of the microscope settings. As oxidative events occur within the macropinosomes, the H2DCFDA signal will become progressively brighter. The TMR signal may remain constant or may vary with the size of macropinosome, as discussed above. The ratio of H2DCFDA:TMR will become progressively larger and will likely plateau within the first 20-30 min in inactivated Raw264.7 cells (Figure 3A,C).
When measuring macropinosomal protein digestion, a progressively strong fluorescent signal will be liberated as the ovalbumin is digested. In Raw264.7 cells, the increase in fluorescence liberated from the digested BODIPY-labeled ovalbumin will plateau within the first 30 min (Figure 3B,D). As before, the TMR signal may remain constant or may vary with size of macropinosome. As degradation of ovalbumin begins immediately after the macropinosome closure, a negative control can be useful in determining the dynamic range of the assay. To this end, acid hydrolases within the macropinosome can be inhibited using an inhibitor of the V-ATPase, such as Concanamycin A (CcA) (Figure 3D) or Bafilomycin A.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62733/62733fig01.jpg" /> 
Figure 1: Principles of dual-fluorophore ratiometric imaging. (A-C). Spectral scans (excitation-fixed, emission-varied) of pH-sensitive fluorophores suspended in buffers of the indicated pH. Fluorescein, pHrodo, and cypHer5e are commonly used as sensors of pH in cellular assays. (D-F). Spectral scans (excitation-fixed, emission-varied) of pH-insensitive fluorophores suspended in buffers of the indicated pH. Dyes that do not vary with pH make useful reference fluorophores. Fluorescein, are not optimally sensitive at the more acidic pH values experienced in endosomes. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62733/62733fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62733/62733fig02.jpg" /> 
Figure 2: Dynamic measurements of macropinosomal pH. (A) Raw264.7 cells were loaded with with fluorescein-labeled 70 kDa dextran and TMR-labeled 70 kDa dextran at 37 &#176;C in the presence or absence of 500 ng/mL of Lipopolysaccharide (LPS) for 15 min. They were also incubated with fluorescein-labeled 70 kDa dextran and TMR-labeled 70 kDa dextran at 4 &#176;C as a control for the non-specific binding of dextran. Insets show individual cells (Scale bars = 20 &#956;m). (B) In situ calibration of Raw264.7 cells loaded with fluorescein-labeled 70 kDa dextran and TMR-labeled 70 kDa dextran using K+-rich buffers at the indicated pH and nigericin. (C) Macropinosome pH in Raw264.7 cells. Insets represent the fluorescein:TMR ratio in individual macropinosomes. Gray boxes indicate the times when cells were being loaded with dextran. Data from two independent experiments each containing &#8805; 80 cells. Data represented as mean &#177; SEM. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62733/62733fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62733/62733fig03.jpg" /> 
Figure 3: Dynamic measurements of oxidative events and protein digestion within the lumen of macropinosomes. (A) Raw264.7 cells were loaded with H2DCFDA-labeled 70 kDa dextran and TMR-labeled 70 kDa dextran at 37 &#176;C for 15 min, followed by a 45 min chase at 37 &#176;C. Insets show ratioed images (H2DCFDA/TMR) of individual macropinosomes. Scale bars = 20 &#956;m. (B) Raw264.7 cells were loaded with BODIPY-labeled ovalbumin and TMR-labeled 70 kDa dextran at 37 &#176;C for 15 min, followed by a 45 min chase at 37 &#176;C. The TMR channel was used to generate a mask, which was applied to the BODIPY channel. Insets show individual cells. Scale bars = 20 &#956;m. (C) H2DCFDA oxidation within the lumen of macropinosomes. Cells were also stimulated with PMA as a positive control. Gray boxes indicate the times when cells were being loaded with dextran. Data from two independent experiments, each containing &#8805; 80 cells. Data represented as mean &#177; SEM. Each time point was compared to the PMA control. A student&#39;s t-test was used for statistical analysis. (D) BODIPY-labeled ovalbumin degradation within macropinosomes. Concanamycin A (CcA) was used as a negative control as it prevents the activation of luminal acid hydrolases. Data from two independent experiments, each containing &#8805; 80 cells. Data represented as mean &#177; SEM. Each time point was compared to the CcA control. A student&#39;s t-test was used for statistical analysis. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62733/62733fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Measuring the pH, Redox Chemistries, and Degradative Capacity of Macropinosomes using Dual-Fluorophore Ratiometric Microscopy at JoVE.com

Measuring the pH, Redox Chemistries, and Degradative Capacity of Macropinosomes using Dual-Fluorophore Ratiometric Microscopy

We describe protocols for measuring pH, oxidative events, and protein digestion in individual macropinosomes in live cells. An emphasis is placed on dual-fluorophore ratiometric microscopy and the advantages it offers over population-based techniques.

In recent years, the field of macropinocytosis has grown rapidly. Macropinocytosis has emerged as a central mechanism by which innate immune cells ...

measuring-ph-redox-chemistries-degradative-capacity-macropinosomes

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

2.3K Views.  University of Victoria. Dit protocol maakt de real-time beoordeling van verschillende processen en biochemische parameters binnen het lumen van individuele macropinosomen mogelijk. Deze methoden kunnen worden gebruikt om geneesmiddelen te ontdekken in de celbiologie van macropinocytose. Dit protocol biedt het voordeel van het onthullen van heterogeniteit op zowel cellulair als organelniveau.Een dag voor het testzaad Raw264.7 cellen met een dichtheid van vijf keer 10 tot de kracht van de cellen in 100 microlit...

Video: Measuring the pH, Redox Chemistries, and Degradative Capacity of Macropinosomes using Dual-Fluorophore Ratiometric Microscopy

Single Liposome Measurements for the Study of Proton-Pumping Membrane Enzymes Using Electrochemistry and Fluorescent Microscopy

Proton-pumping enzymes of electron transfer chains couple redox reactions to proton translocation across the membrane, creating a proton-motive force used for ATP production. The amphiphilic nature of membrane proteins requires particular attention to their handling, and reconstitution into the natural lipid environment is indispensable when studying membrane transport processes like proton translocation. Here, we detail a method that has been used for the investigation of the proton-pumping mechanism of membrane redox enzymes, taking cytochrome bo3 from Escherichia coli as an example. A combination of electrochemistry and fluorescence microscopy is used to control the redox state of the quinone pool and monitor pH changes in the lumen. Due to the spatial resolution of fluorescent microscopy, hundreds of liposomes can be measured simultaneously while the enzyme content can be scaled down to a single enzyme or transporter per liposome. The respective single enzyme analysis can reveal patterns in the enzyme functional dynamics that might be otherwise hidden by the behavior of the whole population. We include a description of a script for automated image analysis.

Here, we present a protocol to study the molecular mechanism of proton translocation across lipid membranes of single liposomes, using cytochrome bo3 as an example. Combining electrochemistry and fluorescence microscopy, pH changes in the lumen of single vesicles, containing single or multiple enzyme, can be detected and analyzed individually.

Proton-pumping enzymes of electron transfer chains couple redox reactions to proton translocation across the membrane, creating a proton-motive force used ...

Biochemistry

Monitoring Changes in Membrane Polarity, Membrane Integrity, and Intracellular Ion Concentrations in Streptococcus pneumoniae Using Fluorescent Dyes

Membrane depolarization and ion fluxes are events that have been studied extensively in biological systems due to their ability to profoundly impact cellular functions, including energetics and signal transductions. While both fluorescent and electrophysiological methods, including electrode usage and patch-clamping, have been well developed for measuring these events in eukaryotic cells, methodology for measuring similar events in microorganisms have proven more challenging to develop given their small size in combination with the more complex outer surface of bacteria shielding the membrane. During our studies of death-initiation in Streptococcus pneumoniae (pneumococcus), we wanted to elucidate the role of membrane events, including changes in polarity, integrity, and intracellular ion concentrations. Searching the literature, we found that very few studies exist. Other investigators had monitored radioisotope uptake or equilibrium to measure ion fluxes and membrane potential and a limited number of studies, mostly in Gram-negative organisms, had seen some success using carbocyanine or oxonol fluorescent dyes to measure membrane potential, or loading bacteria with cell-permeant acetoxymethyl (AM) ester versions of ion-sensitive fluorescent indicator dyes. We therefore established and optimized protocols for measuring membrane potential, rupture, and ion-transport in the Gram-positive organism S. pneumoniae. We developed protocols using the bis-oxonol dye DiBAC4(3) and the cell-impermeant dye propidium iodide to measure membrane depolarization and rupture, respectively, as well as methods to optimally load the pneumococci with the AM esters of the ratiometric dyes Fura-2, PBFI, and BCECF to detect changes in intracellular concentrations of Ca2+, K+, and H+, respectively, using a fluorescence-detection plate reader. These protocols are the first of their kind for the pneumococcus and the majority of these dyes have not been used in any other bacterial species. Though our protocols have been optimized for S. pneumoniae, we believe these approaches should form an excellent starting-point for similar studies in other bacterial species.

Monitoring Changes in Membrane Polarity, Membrane Integrity, and Intracellular Ion Concentrations in Streptococcus pneumoniae Using Fluorescent Dyes

Unlike that seen for eukaryotes, there is a paucity of studies that detail membrane depolarization and ion concentration changes in bacteria, primarily as their small size makes conventional methods of measurement difficult. Here, we detail protocols for monitoring such events in the significant Gram-positive pathogen Streptococcus pneumoniae utilizing fluorescence techniques.

Membrane depolarization and ion fluxes are events that have been studied extensively in biological systems due to their ability to profoundly impact ...

Measuring Phagosome pH by Ratiometric Fluorescence Microscopy

Phagocytosis is a fundamental process through which innate immune cells engulf bacteria, apoptotic cells or other foreign particles in order to kill or neutralize the ingested material, or to present it as antigens and initiate adaptive immune responses. The pH of phagosomes is a critical parameter regulating fission or fusion with endomembranes and activation of proteolytic enzymes, events that allow the phagocytic vacuole to mature into a degradative organelle. In addition, translocation of H+ is required for the production of high levels of reactive oxygen species (ROS), which are essential for efficient killing and signaling to other host tissues. Many intracellular pathogens subvert phagocytic killing by limiting phagosomal acidification, highlighting the importance of pH in phagosome biology. Here we describe a ratiometric method for measuring phagosomal pH in neutrophils using fluorescein isothiocyanate (FITC)-labeled zymosan as phagocytic targets, and live-cell imaging. The assay is based on the fluorescence properties of FITC, which is quenched by acidic pH when excited at 490 nm but not when excited at 440 nm, allowing quantification of a pH-dependent ratio, rather than absolute fluorescence, of a single dye. A detailed protocol for performing in situ dye calibration and conversion of ratio to real pH values is also provided. Single-dye ratiometric methods are generally considered superior to single wavelength or dual-dye pseudo-ratiometric protocols, as they are less sensitive to perturbations such as bleaching, focus changes, laser variations, and uneven labeling, which distort the measured signal. This method can be easily modified to measure pH in other phagocytic cell types, and zymosan can be replaced by any other amine-containing particle, from inert beads to living microorganisms. Finally, this method can be adapted to make use of other fluorescent probes sensitive to different pH ranges or other phagosomal activities, making it a generalized protocol for the functional imaging of phagosomes.

Phagosomal pH influences phagosome maturation, oxidant production, phagosomal killing as well as antigen presentation. Here we describe a ratiometric method for measuring time-course and endpoint pH changes in individual phagosomes in living phagocytes using fluorescence microscopy.

Phagocytosis is a fundamental process through which innate immune cells engulf bacteria, apoptotic cells or other foreign particles in order to kill or ...

Ratiometric Imaging of Extracellular pH in Dental Biofilms

The pH in bacterial biofilms on teeth is of central importance for dental caries, a disease with a high worldwide prevalence. Nutrients and metabolites are not distributed evenly in dental biofilms. A complex interplay of sorption to and reaction with organic matter in the biofilm reduces the diffusion paths of solutes and creates steep gradients of reactive molecules, including organic acids, across the biofilm. Quantitative fluorescent microscopic methods, such as fluorescence life time imaging or pH ratiometry, can be employed to visualize pH in different microenvironments of dental biofilms. pH ratiometry exploits a pH-dependent shift in the fluorescent emission of pH-sensitive dyes. Calculation of the emission ratio at two different wavelengths allows determining local pH in microscopic images, irrespective of the concentration of the dye. Contrary to microelectrodes the technique allows monitoring both vertical and horizontal pH gradients in real-time without mechanically disturbing the biofilm. However, care must be taken to differentiate accurately between extra- and intracellular compartments of the biofilm. Here, the ratiometric dye, seminaphthorhodafluor-4F 5-(and-6) carboxylic acid (C-SNARF-4) is employed to monitor extracellular pH in in vivo grown dental biofilms of unknown species composition. Upon exposure to glucose the dye is up-concentrated inside all bacterial cells in the biofilms; it is thus used both as a universal bacterial stain and as a marker of extracellular pH. After confocal microscopic image acquisition, the bacterial biomass is removed from all pictures using digital image analysis software, which permits to exclusively calculate extracellular pH. pH ratiometry with the ratiometric dye is well-suited to study extracellular pH in thin biofilms of up to 75 &#181;m thickness, but is limited to the pH range between 4.5 and 7.0.

A pH-sensitive ratiometric dye is used in combination with confocal laser scanning microscopy and digital image analysis to monitor extracellular pH in dental biofilms in real-time.

The pH in bacterial biofilms on teeth is of central importance for dental caries, a disease with a high worldwide prevalence. Nutrients and metabolites ...

Imaging the Neutrophil Phagosome and Cytoplasm Using a Ratiometric pH Indicator

Neutrophils are crucial to host innate defense and, consequently, constitute an important area of medical research. The phagosome, the intracellular compartment where the killing and digestion of engulfed particles take place, is the main arena for neutrophil pathogen killing that requires tight regulation. Phagosomal pH is one aspect that is carefully controlled, in turn regulating antimicrobial protease activity. Many fluorescent pH-sensitive dyes have been used to visualize the phagosomal environment. S-1 has several advantages over other pH-sensitive dyes, including its dual emission spectra, its resistance to photo-bleaching, and its high pKa. Using this method, we have demonstrated that the neutrophil phagosome is unusually alkaline in comparison to other phagocytes. By using different biochemical conjugations of the dye, the phagosome can be delineated from the cytoplasm so that changes in the size and shape of the phagosome can be assessed. This allows for further monitoring of ionic movement.

This manuscript describes a simple method to measure the phagosomal pH and area as well as the cytoplasmic pH of human and mouse neutrophils using the ratiometric indicator seminaphthorhodafluor (SNARF)-1, or S-1. This is achieved using live-cell confocal fluorescence microscopy and image analysis.

Neutrophils are crucial to host innate defense and, consequently, constitute an important area of medical research. The phagosome, the intracellular ...

Quantification of Efferocytosis by Single-cell Fluorescence Microscopy

Studying the regulation of efferocytosis requires methods that are able to accurately quantify the uptake of apoptotic cells and to probe the signaling and cellular processes that control efferocytosis. This quantification can be difficult to perform as apoptotic cells are often efferocytosed piecemeal, thus necessitating methods which can accurately delineate between the efferocytosed portion of an apoptotic target versus residual unengulfed cellular fragments. The approach outlined herein utilizes dual-labeling approaches to accurately quantify the dynamics of efferocytosis and efferocytic capacity of efferocytes such as macrophages. The cytosol of the apoptotic cell is labeled with a cell-tracking dye to enable monitoring of all apoptotic cell-derived materials, while surface biotinylation of the apoptotic cell allows for differentiation between internalized and non-internalized apoptotic cell fractions. The efferocytic capacity of efferocytes is determined by taking fluorescent images of live or fixed cells and quantifying the amount of bound versus internalized targets, as differentiated by streptavidin staining. This approach offers several advantages over methods such as flow cytometry, namely the accurate delineation of non-efferocytosed versus efferocytosed apoptotic cell fractions, the ability to measure efferocytic dynamics by live-cell microscopy, and the capacity to perform studies of cellular signaling in cells expressing fluorescently-labeled transgenes. Combined, the methods outlined in this protocol serve as the basis for a flexible experimental approach that can be used to accurately quantify efferocytic activity and interrogate cellular signaling pathways active during efferocytosis.

Efferocytosis, the phagocytic removal of apoptotic cells, is required to maintain homeostasis and is facilitated by receptors and signaling pathways that allow for the recognition, engulfment, and internalization of apoptotic cells. Herein, we present a fluorescence microscopy protocol for the quantification of efferocytosis and the activity of efferocytic signaling pathways.

Studying the regulation of efferocytosis requires methods that are able to accurately quantify the uptake of apoptotic cells and to probe the signaling ...

Monitoring Extracellular pH in Cross-Kingdom Biofilms using Confocal Microscopy

Cross-kingdom biofilms consisting of both fungal and bacterial cells are involved in a variety of oral diseases, such as endodontic infections, periodontitis, mucosal infections and, most notably, early childhood caries. In all of these conditions, the pH in the biofilm matrix impacts microbe-host interactions and thus the disease progression. The present protocol describes a confocal microscopy-based method to monitor pH dynamics inside cross-kingdom biofilms comprising Candida albicans and Streptococcus mutans. The pH-dependent dual-emission spectrum and the staining properties of the ratiometric probe C-SNARF-4 are exploited to determine drops in pH in extracellular areas of the biofilms. Use of pH ratiometry with the probe requires a meticulous choice of imaging parameters, a thorough calibration of the dye, and careful, threshold-based post-processing of the image data. When used correctly, the technique allows for the rapid assessment of extracellular pH in different areas of a biofilm and thus the monitoring of both horizontal and vertical pH gradients over time. While the use of confocal microscopy limits Z-profiling to thin biofilms of 75 &#181;m or less, the use of pH ratiometry is ideally suited for the noninvasive study of an important virulence factor in cross-kingdom biofilms.

The protocol describes the cultivation of cross-kingdom biofilms consisting of Candida albicans and Streptococcus mutans and presents a confocal microscopy-based method for the monitoring of extracellular pH inside these biofilms.

Cross-kingdom biofilms consisting of both fungal and bacterial cells are involved in a variety of oral diseases, such as endodontic infections, ...

Measurement of Vacuolar and Cytosolic pH In Vivo in Yeast Cell Suspensions

Vacuolar and cytosolic pH are highly regulated in yeast cells and occupy a central role in overall pH homeostasis. We describe protocols for ratiometric measurement of pH in vivo using pH-sensitive fluorophores localized to the vacuole or cytosol. Vacuolar pH is measured using BCECF, which localizes to the vacuole in yeast when introduced into cells in its acetoxymethyl ester form. Cytosolic pH is measured with a pH-sensitive GFP expressed under control of a yeast promoter, yeast pHluorin. Methods for measurement of fluorescence ratios in yeast cell suspensions in a fluorimeter are described. Through these protocols, single time point measurements of pH under different conditions or in different yeast mutants have been compared and changes in pH over time have been monitored. These methods have also been adapted to a fluorescence plate reader format for high-throughput experiments. Advantages of ratiometric pH measurements over other approaches currently in use, potential experimental problems and solutions, and prospects for future use of these techniques are also described.

Measurement of Vacuolar and Cytosolic pH In Vivo in Yeast Cell Suspensions

Vacuolar and cytosolic pH can be measured in live yeast (S. cerevisiae) cells using ratiometric fluorescent dyes localized to specific cellular compartments. We describe procedures for measuring vacuolar pH with BCECF-AM, which localizes to the vacuole in yeast, and cytosolic pH with a cytosolic ratiometric pH-sensitive GFP (yeast pHluorin).

Vacuolar and  cytosolic pH are highly regulated in yeast cells and occupy a central role in  overall pH homeostasis. We describe  protocols for ...

Biology

Simultaneous pH Measurement in Endocytic and Cytosolic Compartments in Living Cells using Confocal Microscopy

Intracellular pH is tightly regulated and differences in pH between the cytoplasm and organelles have been reported1. Regulation of cellular pH is crucial for homeostatic control of physiological processes that include: protein, DNA and RNA synthesis, vesicular trafficking, cell growth and cell division. Alterations in cellular pH homeostasis can lead to detrimental functional changes and promote progression of various diseases2. Various methods are available for measuring intracellular pH but very few of these allow simultaneous measurement of pH in the cytoplasm and in organelles. Here, we describe in detail a rapid and accurate method for the simultaneous measurement of cytoplasmic and organellar pH by using confocal microscopy on living cells3. This goal is achieved with the use of two pH-sensing ratiometric dyes that possess selective cellular compartment partitioning. For instance, SNARF-1 is compartmentalized inside the cytoplasm whereas HPTS is compartmentalized inside endosomal/lysosomal organelles. Although HPTS is commonly used as a cytoplasmic pH indicator, this dye can specifically label vesicles along the endosomal-lysosomal pathway after being taken up by pinocytosis3,4. Using these pH-sensing probes, it is possible to simultaneously measure pH within the endocytic and cytoplasmic compartments. The optimal excitation wavelength of HPTS varies depending on the pH while for SNARF-1, it is the optimal emission wavelength that varies. Following loading with SNARF-1 and HPTS, cells are cultured in different pH-calibrated solutions to construct a pH standard curve for each probe. Cell imaging by confocal microscopy allows elimination of artifacts and background noise. Because of the spectral properties of HPTS, this probe is better suited for measurement of the mildly acidic endosomal compartment or to demonstrate alkalinization of the endosomal/lysosomal organelles. This method simplifies data analysis, improves accuracy of pH measurements and can be used to address fundamental questions related to pH modulation during cell responses to external challenges.

Several methods are available for measuring intracellular pH but very few of these allow simultaneous measurement of cytoplasmic and organellar pH. Here, we describe in detail a rapid and accurate methodology to simultaneously measure cytoplasmic and vesicular pH by ratiometric imaging of living cells.

Intracellular pH is tightly regulated and differences in pH between the cytoplasm and organelles have been reported1. Regulation of cellular pH is crucial ...

Optical Quantification of Intracellular pH in Drosophila melanogaster Malpighian Tubule Epithelia with a Fluorescent Genetically-encoded pH Indicator

Epithelial ion transport is vital to systemic ion homeostasis as well as maintenance of essential cellular electrochemical gradients. Intracellular pH (pHi) is influenced by many ion transporters and thus monitoring pHi is a useful tool for assessing transporter activity. Modern Genetically Encoded pH-Indicators (GEpHIs) provide optical quantification of pHi in intact cells on a cellular and subcellular scale. This protocol describes real-time quantification of cellular pHi regulation in Malpighian Tubules (MTs) of Drosophila melanogaster through ex vivo live-imaging of pHerry, a pseudo-ratiometric GEpHI with a pKa well-suited to track pH changes in the cytosol. Extracted adult fly MTs are composed of morphologically and functionally distinct sections of single-cell layer epithelia, and can serve as an accessible and genetically tractable model for investigation of epithelial transport. GEpHIs offer several advantages over conventional pH-sensitive fluorescent dyes and ion-selective electrodes. GEpHIs can label distinct cell populations provided appropriate promoter elements are available. This labeling is particularly useful in ex vivo, in vivo, and in situ preparations, which are inherently heterogeneous. GEpHIs also permit quantification of pHi in intact tissues over time without need for repeated dye treatment or tissue externalization. The primary drawback of current GEpHIs is the tendency to aggregate in cytosolic inclusions in response to tissue damage and construct over-expression. These shortcomings, their solutions, and the inherent advantages of GEpHIs are demonstrated in this protocol through assessment of basolateral proton (H+) transport in functionally distinct principal and stellate cells of extracted fly MTs. The techniques and analysis described are readily adaptable to a wide variety of vertebrate and invertebrate preparations, and the sophistication of the assay can be scaled from teaching labs to intricate determination of ion flux via specific transporters.

Optical Quantification of Intracellular pH in Drosophila melanogaster Malpighian Tubule Epithelia with a Fluorescent Genetically-encoded pH Indicator

Cellular ion transport can often be assessed by monitoring intracellular pH (pHi). Genetically Encoded pH-Indicators (GEpHIs)&#160;provide optical quantification of intracellular pH in intact cells. This protocol details the quantification of intracellular pH through cellular ex vivo live-imaging of Malpighian tubules of Drosophila melanogaster with pHerry, a pseudo-ratiometric genetically encoded pH-indicator.

Epithelial ion transport is vital to systemic ion homeostasis as well as maintenance of essential cellular electrochemical gradients. Intracellular pH ...

Measuring the pH, Redox Chemistries, and Degradative Capacity of Macropinosomes using Dual-Fluorophore Ratiometric Microscopy

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