Name: automated imaging and analysis for the quantification of fluorescently labeled macropinosomes
Uploaded: 2021-08-24T15:00:00
Description: Watch this Scientific Journal Video about Automated Imaging and Analysis for the Quantification of Fluorescently Labeled Macropinosomes at JoVE.com

This work was supported by NIH/NCI grants (R01CA207189, R21CA243701) to C.C. KMO.G. is a recipient of a TRDRP Postdoctoral Fellowship Award (T30FT0952). The BioTek Cytation 5 is a part of the Sanford Burnham Prebys Cell Imaging Core, which receives financial support from the NCI Cancer Center Support Grant (P30 CA030199). Figures 1-3 were created using BioRender.

1. Preparation of materials
<ol>
	<li>Dissolve 70 kDa dextran labeled with FITC or tetramethylrhodamine (TMR) in PBS to obtain a 20 mg/mL solution. Store the aliquots at -20 &#176;C.</li>
	<li>Dissolve DAPI in ddH2O to obtain a 1 mg/mL solution. Store the aliquots at -20 &#176;C. 
	CAUTION: DAPI is a potential carcinogen and should be handled with care.</li>
	<li>On the day of fixation, prepare fresh 3.7% ACS grade formaldehyde in PBS. 
	CAUTION: Formaldehyde is a fixative, known carcinogen and is ...

<ol>
	<li>Lin, X. P., Mintern, J. D., Gleeson, 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+different+cell+types:+similarities+and+differences.">Macropinocytosis in different cell types: similarities and differences.</a> Membranes. 10 (8), 21 (2020).</li><li>Recouvreux, M. V., 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=Macropinocytosis:+a+metabolic+adaptation+to+nutrient+stress+in+cancer.">Macropinocytosis: a metabolic adaptation to nutrient stress in cancer.</a> Frontiers in Endocrinology. 8, (2017).</li><li>Zhang, Y. J., 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=Macropinocytosis+in+cancer:+a+complex+signaling+network.">Macropinocytosis in cancer: a complex signaling network.</a> Trends in Cancer. 5 (6), 332-334 (2019).</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 (7451), 633-637 (2013).</li><li>Kim, 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=PTEN+deficiency+and+AMPK+activation+promote+nutrient+scavenging+and+anabolism+in+prostate+cancer+cells.">PTEN deficiency and AMPK activation promote nutrient scavenging and anabolism in prostate cancer cells.</a> Cancer Discovery. 8 (7), 866-883 (2018).</li><li>Jayashankar, V., Edinger, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macropinocytosis+confers+resistance+to+therapies+targeting+cancer+anabolism.">Macropinocytosis confers resistance to therapies targeting cancer anabolism.</a> Nature Communications. 11 (1), (2020).</li><li>Kamphorst, J. 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=Human+pancreatic+cancer+tumors+are+nutrient+poor+and+tumor+cells+actively+scavenge+extracellular+protein.">Human pancreatic cancer tumors are nutrient poor and tumor cells actively scavenge extracellular protein.</a> Cancer Research. 75 (3), 544-553 (2015).</li><li>Olivares, O., 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=Collagen-derived+proline+promotes+pancreatic+ductal+adenocarcinoma+cell+survival+under+nutrient+limited+conditions.">Collagen-derived proline promotes pancreatic ductal adenocarcinoma cell survival under nutrient limited conditions.</a> Nature Communications. 8, (2017).</li><li>Seguin, 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=Galectin-3,+a+druggable+vulnerability+for+KRAS-addicted+cancers.">Galectin-3, a druggable vulnerability for KRAS-addicted cancers.</a> Cancer Discovery. 7 (12), 1464-1479 (2017).</li><li>Redelman-Sidi, 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=The+canonical+Wnt+pathway+drives+macropinocytosis+in+cancer.">The canonical Wnt pathway drives macropinocytosis in cancer.</a> Cancer Research. 78 (16), 4658-4670 (2018).</li><li>Tejeda-Munoz, N., Albrecht, L. V., Bui, M. H., De Robertis, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wnt+canonical+pathway+activates+macropinocytosis+and+lysosomal+degradation+of+extracellular+proteins.">Wnt canonical pathway activates macropinocytosis and lysosomal degradation of extracellular proteins.</a> Proceedings of the National Academy of Sciences of the United States of America. 116 (21), 10402-10411 (2019).</li><li>Schmees, 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+the+PDGF+beta-receptor+promotes+fibroblast+transformation+by+H-RasG12V.">Macropinocytosis of the PDGF beta-receptor promotes fibroblast transformation by H-RasG12V.</a> Molecular Biology of the Cell. 23 (13), 2571-2582 (2012).</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 (3), 381-392 (2019).</li><li>Recouvreux, M. V., 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=Glutamine+depletion+regulates+Slug+to+promote+EMT+and+metastasis+in+pancreatic+cancer.">Glutamine depletion regulates Slug to promote EMT and metastasis in pancreatic cancer.</a> Journal of Experimental Medicine. 217 (9), (2020).</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. 11 (7), 1808-1825 (2021).</li><li>Su, 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=Cancer+cells+escape+autophagy+inhibition+via+NRF2-induced+macropinocytosis.">Cancer cells escape autophagy inhibition via NRF2-induced macropinocytosis.</a> Cancer Cell. 39 (5), 678-693 (2021).</li><li>Bar-Sagi, D., Feramisco, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+membrane+ruffling+and+fluid-phase+pinocytosis+in+quiescent+fibroblasts+by+ras+proteins.">Induction of membrane ruffling and fluid-phase pinocytosis in quiescent fibroblasts by ras proteins.</a> Science. 233 (4768), 1061-1068 (1986).</li><li>Mishra, 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=Stromal+epigenetic+alterations+drive+metabolic+and+neuroendocrine+prostate+cancer+reprogramming.">Stromal epigenetic alterations drive metabolic and neuroendocrine prostate cancer reprogramming.</a> Journal of Clinical Investigation. 128 (10), 4472-4484 (2018).</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 (1), 182-192 (2014).</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>Wang, J. T. H., Teasdale, R. D., Liebl, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macropinosome+quantitation+assay.">Macropinosome quantitation assay.</a> MethodsX. 1, 36-41 (2014).</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>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></ol>

The quality of the experiments and data acquisition highly depends on the quality of the reagents, the optimization of the settings, and the cleanliness of the coverslips and microplate. The final results should give minimal variation between replicates; however, biological variations do naturally occur or may otherwise be caused by a number of factors. Cell density may cause cells to respond more or less to macropinocytosis inducers or inhibitors. It is, therefore, crucial to adhere to the 80% confluency as proposed her...

The non-specific endocytic pathway of macropinocytosis allows cells to internalize a variety of extracellular components, including nutrients, proteins, antigens, and pathogens, through bulk uptake of extracellular fluid and its constituents1. Though important for the biology of numerous cell types, increasingly, the macropinocytosis pathway is described to play an essential role in tumor biology, where, through macropinocytic uptake, tumor cells are able to survive and proliferate in the presence of a nutrient-depleted microenvironment2,3. The uptake of extracellular macromolecules, including albumin and extracellular matrix, and necrotic cell debris, provides an alternative nutrient source for biomass production by creating amino acids, sugars, lipids and nucleotides through macropinosome and lysosome fusion-mediated cargo catabolism4,5,6,7,8.
The induction and regulation of macropinocytosis are complex and can vary depending on cellular context. Thus far, several inducers of macropinocytosis have been identified and include ligands, such as epidermal growth factor (EGF), platelet-derived growth factor (PDGF), galectin-3, and Wnt3A9,10,11,12,13. In addition, culturing conditions that mimic the tumor microenvironment can trigger activation of the pathway. Pancreatic ductal adenocarcinoma (PDAC) tumors are nutrient-deprived, especially for the amino acid glutamine, which causes both cancer cells and cancer-associated fibroblasts (CAFs) to rely on macropinocytosis for survival7,13,14,15. Moreover, tumor stresses, such as hypoxia and oxidative stress, can activate this scavenging pathway16. In addition to the numerous extrinsic influencers that can induce macropinocytosis, a variety of intracellular pathways control macropinosome formation. Oncogenic Ras-mediated transformation is sufficient to initiate the macropinocytic machinery, and multiple cancer types exhibit oncogenic Ras-driven constitutive macropinocytosis4,5,9,17. Alternatively, wild-type Ras activation and Ras-independent pathways have been identified to activate macropinocytosis in cancer cells and CAFs10,11,15,18. The use of various in vitro models in combination with inhibitor treatments has resulted in the identification of several macropinocytosis modulators, which include sodium-hydrogen exchangers, the small GTPase Rac1, phosphoinositide 3-kinase (PI3K), p21-activated kinase (Pak), and AMP-activated protein kinase (AMPK)4,13,15. However, given the multitude of described factors and conditions that regulate macropinocytosis, it is conceivable that many more modulators and stimuli remain undiscovered. The identification of novel modulators and stimuli can be facilitated by automated assessment of a multitude of conditions in a single experiment. This methodology can shed light on the factors involved in macropinosome formation and may allow for the discovery of novel small molecules or biologics that target this pathway.
Here, we have adapted our previously established protocol for determining the extent of macropinocytosis in cancer cells in vitro to a 96-well microplate format and automated imaging and quantification19,20. This protocol is based on fluorescent microscopy, which has become a standard in the field to determine macropinocytosis in vitro and in vivo4,5,6,7,9,10,11,12,13,15,16,17,18,19,20,21,22. Macropinosomes can be distinguished from other endocytic pathways through their ability to internalize large macromolecules, such as high molecular weight dextran (i.e., 70 kDa)2,3,4,20,21,22,23. Thus, macropinosomes can be defined through uptake of extracellularly administered fluorophore-labeled 70 kDa dextran. As a result, macropinocytic vesicles manifest as intracellular clusters of fluorescent puncta with sizes ranging from 0.2-5 &#181;m. These puncta can be microscopically imaged and subsequently quantified to determine the extent of macropinocytosis in the cell - &#39;the macropinocytic index&#39;.
In this protocol, the essential steps to visualize macropinosomes in adherent cells in vitro on a 96-well microplate and coverslips using standard laboratory equipment are described (Figure 1). In addition, the directions to automate the image acquisition and quantification of the macropinocytic index using a cell imaging multi-mode plate reader are provided. This automation reduces time, cost, and effort compared to our previously described protocols19,20. In addition, it avoids unintentionally biased imaging acquisition and analysis and thereby enhances reproducibility and reliability. This method can easily be adapted to different cell types or plate readers or be utilized to determine alternative macropinosome features, such as size, number, and location. The herein described method is especially suitable for the screening of cell culture conditions that induce macropinocytosis, the identification of novel modulators, or optimization of drug concentrations of known inhibitors.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62828/62828fig01.jpg" /> 
Figure 1: Schematic of the automated assay to determine the &#39;macropinocytic index&#39; in adherent cells. Created using BioRender. <a href="https://www.jove.com/files/ftp_upload/62828/62828fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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	<tbody>
		<tr>
			<td>0.25% Trypsin</td>
			<td>Corning</td>
			<td>25053CI</td>
			<td>0.1% EDTA in HBSS w/o Calcium, Magnesium and Sodium Bicarbonate</td>
		</tr>
		<tr>
			<td>1.5 mL Microcentrifuge tube</td>
			<td>Fisherbrand</td>
			<td>05-408-129</td>
			<td></td>
		</tr>
		<tr>
			<td>10-cm Tissue culture dish</td>
			<td>Greiner Bio-One</td>
			<td>664160</td>
			<td>CELLSTAR</td>
		</tr>
		<tr>
			<td>15 mL Centrifuge tube</td>
			<td>Fisherbrand</td>
			<td>07-200-886</td>
			<td></td>
		</tr>
		<tr>
			<td>2 L Beaker</td>
			<td>Fisherbrand</td>
			<td>02-591-33</td>
			<td></td>
		</tr>
		<tr>
			<td>24-well Tissue culture plate</td>
			<td>Greiner Bio-One</td>
			<td>662160</td>
			<td>CELLSTAR</td>
		</tr>
		<tr>
			<td>25 mL Reagent reservoir</td>
			<td>Genesee Scientific Corporation</td>
			<td>28-121</td>
			<td></td>
		</tr>
		<tr>
			<td>500 mL Beaker</td>
			<td>Fisherbrand</td>
			<td>02-591-30</td>
			<td></td>
		</tr>
		<tr>
			<td>6-cm Tissue culture dish</td>
			<td>Greiner Bio-One</td>
			<td>628160</td>
			<td>CELLSTAR</td>
		</tr>
		<tr>
			<td>8-Channel aspiration adapter</td>
			<td>Integra Biosciences</td>
			<td>155503</td>
			<td></td>
		</tr>
		<tr>
			<td>8-Channel aspiration adapter for standard tips</td>
			<td>Integra Biosciences</td>
			<td>159024</td>
			<td></td>
		</tr>
		<tr>
			<td>95% Ethanol</td>
			<td>Decon Laboratories Inc</td>
			<td>4355226</td>
			<td></td>
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			<td>Ammonia-free glass cleaner</td>
			<td>Sparkle</td>
			<td>FUN20500CT</td>
			<td></td>
		</tr>
		<tr>
			<td>Black 96-well high-content screening microplate</td>
			<td>PerkinElmer</td>
			<td>6055300</td>
			<td>CellCarrier-96 Ultra</td>
		</tr>
		<tr>
			<td>Cotton-tipped applicator</td>
			<td>Fisherbrand</td>
			<td>23-400-101</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslips</td>
			<td>Fisherbrand</td>
			<td>12-545-80</td>
			<td>12 mm diameter</td>
		</tr>
		<tr>
			<td>Cytation 5 Cell Imaging Multi-Mode Reader</td>
			<td>Biotek</td>
			<td>CYT5FW</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Millipore Sigma</td>
			<td>5.08741</td>
			<td></td>
		</tr>
		<tr>
			<td>Dextran 70 kDa - FITC</td>
			<td>Life Technologies</td>
			<td>D1822</td>
			<td>Lysine-fixable</td>
		</tr>
		<tr>
			<td>Dextran 70 kDa - TMR</td>
			<td>Life Technologies</td>
			<td>D1819</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Millipore Sigma</td>
			<td>D1435</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS</td>
			<td>Corning</td>
			<td>21031CV</td>
			<td>Without Calcium and Magnesium</td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Fine Science Tools</td>
			<td>11251-20</td>
			<td>Dumont #5</td>
		</tr>
		<tr>
			<td>Formaldehyde, 37%</td>
			<td>Ricca Chemical</td>
			<td>RSOF0010-250A</td>
			<td>ACS Reagent Grade</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Fisher BioReagents</td>
			<td>BP229-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Hardening fluorescence mounting media</td>
			<td>Agilent Tech</td>
			<td>S302380-2</td>
			<td>DAKO</td>
		</tr>
		<tr>
			<td>Hoechst 33342</td>
			<td>Millipore Sigma</td>
			<td>B2261</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric acid (HCl)</td>
			<td>Fisher Chemical</td>
			<td>A144-212</td>
			<td>Certified ACS Plus, 36.5%&#8211;38.0%</td>
		</tr>
		<tr>
			<td>Lint-free wipes</td>
			<td>Kimberly-Clark</td>
			<td>34155</td>
			<td>Kimwipes</td>
		</tr>
		<tr>
			<td>Miscroscope slides</td>
			<td>Fisherbrand</td>
			<td>12-544-1</td>
			<td>Premium plain glass</td>
		</tr>
		<tr>
			<td>Multichannel pipette</td>
			<td>Gilson</td>
			<td>FA10013</td>
			<td>8 channels, 0.5&#8211;10 &#181;L</td>
		</tr>
		<tr>
			<td>Multichannel pipette</td>
			<td>Gilson</td>
			<td>FA10012&#160;</td>
			<td>12 channels, 20&#8211;200 &#181;L</td>
		</tr>
		<tr>
			<td>Multichannel pipette</td>
			<td>Gilson</td>
			<td>FA10011</td>
			<td>8 channels, 20&#8211;200 &#181;L</td>
		</tr>
		<tr>
			<td>Parafilm M</td>
			<td>Pechiney</td>
			<td>PM996</td>
			<td></td>
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		<tr>
			<td>Plastic wrap</td>
			<td>Kirkland Signature</td>
			<td>208733</td>
			<td>Stretch-Tite</td>
		</tr>
		<tr>
			<td>Silicone isolators</td>
			<td>Grace Bio Labs Inc</td>
			<td>664107</td>
			<td>13 mm Diameter X 0.8 mm Depth ID, 25 mm X 25 mm</td>
		</tr>
		<tr>
			<td>Slide adapter</td>
			<td>Biotek</td>
			<td>1220548</td>
			<td></td>
		</tr>
		<tr>
			<td>Wash bottle</td>
			<td>Fisherbrand</td>
			<td>FB0340922C</td>
			<td></td>
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</table>

automated imaging and analysis for the quantification of fluorescently labeled macropinosomes

Macropinocytosis is a non-specific fluid-phase uptake pathway that allows cells to internalize large extracellular cargo, such as proteins, pathogens, and cell debris, through bulk endocytosis. This pathway plays an essential role in a variety of cellular processes, including the regulation of immune responses and cancer cell metabolism. Given this importance in biological function, examining cell culture conditions can provide valuable information by identifying regulators of this pathway and optimizing conditions to be employed in the discovery of novel therapeutic approaches. The study describes an automated imaging and analysis technique using standard laboratory equipment and a cell imaging multi-mode plate reader for the rapid quantification of the macropinocytic index in adherent cells. The automated method is based on the uptake of high molecular weight fluorescent dextran and can be applied to 96-well microplates to facilitate assessments of multiple conditions in one experiment or fixed samples mounted onto glass coverslips. This approach is aimed at maximizing reproducibility and reducing experimental variation while being both time-saving and cost-effective.

When the steps and adjustment of the above-described protocol are followed accordingly, the final experimental results should provide information about whether the studied cell culture conditions or inhibitors induce or reduce macropinocytosis in the cell line of interest. To strengthen the validity of these findings, the inclusion of control conditions will allow for the scrutinization of the results to determine whether the experiment has been completed successfully. Macropinocytosis induction controls will provide inf...

Watch this Scientific Journal Video about Automated Imaging and Analysis for the Quantification of Fluorescently Labeled Macropinosomes at JoVE.com

Automated Imaging and Analysis for the Quantification of Fluorescently Labeled Macropinosomes

Automated assays using multi-well microplates are advantageous approaches for identifying pathway regulators by allowing the assessment of a multitude of conditions in a single experiment. Here, we have adapted the well-established macropinosome imaging and quantification protocol to a 96-well microplate format and provide a comprehensive outline for automation using a multi-mode plate reader.

Macropinocytosis is a non-specific fluid-phase uptake pathway that allows cells to internalize large extracellular cargo, such as proteins, pathogens, and ...

Macropinocytosis is an endocytic pathway that is important for a variety of cellular processes, including cancer metabolism. This protocol allows you to quantify the extent of macropinocytosis in cells in vitro. The automation is aimed at maximizing reproducibility, productivity and reducing experimental variation.Moreover, the fluorescent microscopy allows you to visually inspect the sample to determine experimental quality and assess additional macropinosome characteristics. Demonstrating the procedure with me will be Cheska Marie Galapate, a research assistant from my laboratory. For the 24-well plate with coverslip format, use forceps to grip a single coverslip from the ethanol bath.Tap the coverslip to the inside wall of the plate to remove excess ethanol and place the coverslip flat on the bottom of the well. After the ethanol evaporates, wash the coverslip twice with DPBS, then seed the cells on top of the coverslip by adding 500 microliters of the cell suspension to each well. Place the cells in a 37 degree Celsius cell incubator with 5%carbon dioxide until cell confluency reaches 60 to 80%on the day before macropinosome labeling.The day before macropinosome labeling, replace the media in the wells with 500 microliters of pre-warmed serum-free media and place the cells in the incubator for 16 to 24 hours. For the 96-well microplate format, begin by transferring the cell suspension to a 25 milliliter reagent reservoir. Then using a multichannel pipette, seed 100 microliters of the cell suspension to each well of a black 96-well high-content screening microplate with an optically clear cyclic olefin or glass bottom.Incubate the cells until cell confluency reaches 60 to 80%on the day before macropinosome labeling. The day before macropinosome labeling, remove and discard the media from each well using a multichannel aspiration adaptor for standard tips attached to a vacuum pump. Alternatively, a multichannel pipette can be used.Using a reagent reservoir and multichannel pipette, gently add 100 microliters of pre-warmed serum-free media to each well, then place the cells in the cell incubator for 16 to 24 hours. For the 24-well plate with coverslip format, replace the media in the wells with 200 microliters of serum-free media with fluorophore-labeled high molecular weight dextran and place the cells in the cell incubator for 30 minutes. After the incubation, aspirate the media and gently but quickly wash the cells five times with ice cold PBS using a pre-cooled wash bottle.Firmly shake the plate by hand during washes to aid in dislodging dextran aggregates that become stuck to the coverslips. Next, fix the cells by adding 350 microliters of 3.7%formaldehyde and incubating for 20 minutes, then aspirate the fixation solution and wash the cells with PBS twice. Stain the nuclei with 350 microliters of DAPI in PBS.After 20 minutes, aspirate the DAPI solution and wash the cells with PBS thrice. Adhere silicone isolators side by side on a microscope slide to obtain even spacing and reproducible localization of the coverslips required for imaging automation. Then for each coverslip, add a drop of hardening fluorescence mounting media on the microscope slide within the open space of the isolator.Pick up a coverslip using forceps and remove excess PBS by gently tapping the side of the coverslips on a lint-free wipe. Next, place the coverslip upside down on the drop of mounting media and gently tap the coverslip using closed forceps to remove bubbles from the mounting media. Store the slides in a dark environment and allow the mounting media to dry at room temperature, typically taking 16 to 24 hours.Before imaging, remove the isolators from the microscope slide. After the slides have been equilibrated to room temperature, clean the coverslips using a cotton tipped applicator wetted with ammonia-free glass cleaner. Subsequently, use a clean cotton tipped applicator wetted with 70%ethanol to clean the coverslip and leave it dry.For the 96-well microplate format, after aspirating the wells as demonstrated previously, add 40 microliters of serum-free media with fluorophore-labeled high molecular weight dextran to the wells, then incubate the cells in the cell incubator for 30 minutes. After incubation, discard the media in the microplate by manually flicking the plate upside down into an empty five liter beaker, then rinse the cells and the microplate twice by slowly submerging the plate vertically at a slight angle into a two liter beaker filled with ice cold PBS and subsequently discarding the PBS in the microplate by flicking the plate upside down into the five liter beaker. After the last PBS rinse, fix the cells by adding 100 microliters of 3.7%formaldehyde in PBS to each well using a 25 milliliter reagent reservoir and a multichannel pipette.After a 20-minute incubation at room temperature, remove the fixation solution and wash the cells with PBS using the submerging and flicking technique. After the second PBS wash, stain the nuclei with 100 microliters of DAPI in PBS per well. After 20 minutes, rinse the cells thrice with ice cold PBS as demonstrated previously.Remove any residual PBS by tapping the microplate upside down onto a lint-free wipe. Then add 100 microliters of fresh PBS to each well using a 25 milliliter reagent reservoir and multichannel pipette. Before imaging, let the plate equilibrate to room temperature, then wipe the cell culture plate dry with a lint-free wipe.Alternatively, store the plate covered away from light at four degrees Celsius for up to one week. For automated macropinosome imaging, create an automation protocol to acquire the images with a 40X air objective in the wavelength channel of the dextran fluorophore and DAPI. Next, optimize exposure settings using a sample predicted to have the highest level of macropinocytosis to avoid over-exposure, which may result in saturation of the signal and loss of intensity data.Use focus settings that readily and consistently locate the sample to produce high-quality images. Acquire multiple images across each well or coverslip to account for sample variability and obtain an accurate representation of the sample. To determine the macropinocytic index, subtract the background for the DAPI and corresponding dextran image by applying the appropriate function, frequently called the rolling ball function.Adjust the settings so that the background noise is minimized with minimal to no subtraction effect on the DAPI and dextran signal. Next, using a field with high dextran signal, determine the intensity signal settings, frequently called the threshold function, to select the nuclei, then determine the minimum intensity signal setting required to select only the macropinosomes. For the dextran image, calculate the total fluorescence within the created macropinosome selection or use the selection to determine the total area positive for dextran.For the DAPI image, use the selection to determine the number of nuclei in the image to reflect the number of cells present. Then determine the macropinocytic index by dividing the total dextran fluorescence or area by the number of cells determined by DAPI. In AsPC-1 PDAK cells, adding EGF at 100 nanograms per milliliter for five minutes before adding the dextran activates macropinocytosis.Moreover, autocrine EGF activation of macropinocytosis can be induced by glutamine deprivation for 16 to 24 hours. MIA PaCa-2 cells show constitutive macropinocytosis which is inhibited by a 30-minute treatment with 75 micromolar EIPA or a two-hour treatment with 10 micromolar EHop-016. In a dose response experiment, the macropinocytic index gradually decreased at higher drug concentrations of EHop-016 and EIPA, thereby confirming the existence of constitutive Rac1-dependent macropinocytosis.You can adapt this procedure to assess macropinocytosis of other fluorescently tagged cargo such as albumin. In addition, it is recommended to evaluate whether macropinocytic uptake of albumin contributes to cell fitness by performing cell viability assays. This technique has been central to the identification of macropinocytic nutrient supply in cancer and stromal cells and the automation has greatly increased our condition testing capacity.

Research

JoVE Journal

Biology

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