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.

<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>

C.C. is an inventor on an issued patent titled ''Cancer diagnostics, therapeutics, and drug discovery associated with macropinocytosis,'' Patent No.: 9,983,194.

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>

<table><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></tr><tr><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%&amp;ndash;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&amp;ndash;10 &amp;micro;L</td></tr><tr><td>Multichannel pipette</td><td>Gilson</td><td>FA10012&amp;nbsp;</td><td>12 channels, 20&amp;ndash;200 &amp;micro;L</td></tr><tr><td>Multichannel pipette</td><td>Gilson</td><td>FA10011</td><td>8 channels, 20&amp;ndash;200 &amp;micro;L</td></tr><tr><td>Parafilm M</td><td>Pechiney</td><td>PM996</td><td></td></tr><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></tr></tbody></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 ...

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Research

JoVE Journal

Biology

2.6K Views.  NCI-designated Cancer Center. 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.

Video: Automated Imaging and Analysis for the Quantification of Fluorescently Labeled Macropinosomes

Study of Phagolysosome Biogenesis in Live Macrophages

Phagocytic cells play a major role in the innate immune system by removing and eliminating invading microorganisms in their phagosomes. Phagosome maturation is the complex and tightly regulated process during which a nascent phagosome undergoes drastic transformation through well-orchestrated interactions with various cellular organelles and compartments in the cytoplasm. This process, which is essential for the physiological function of phagocytic cells by endowing phagosomes with their lytic and bactericidal properties, culminates in fusion of phagosomes with lysosomes and biogenesis of phagolysosomes which is considered to be the last and critical stage of maturation for phagosomes. In this report, we describe a live cell imaging based method for qualitative and quantitative analysis of the dynamic process of lysosome to phagosome content delivery, which is a hallmark of phagolysosome biogenesis. This approach uses IgG-coated microbeads as a model for phagocytosis and fluorophore-conjugated dextran molecules as a luminal lysosomal cargo probe, in order to follow the dynamic delivery of lysosmal content to the phagosomes in real time in live macrophages using time-lapse imaging and confocal laser scanning microscopy. Here we describe in detail the background, the preparation steps and the step-by-step experimental setup to enable easy and precise deployment of this method in other labs. Our described method is simple, robust, and most importantly, can be easily adapted to study phagosomal interactions and maturation in different systems and under various experimental settings such as use of various phagocytic cells types, loss-of-function experiments, different probes, and phagocytic particles.

Phagocytic cells play a major role in the innate immune system by removing and eliminating invading microorganisms in their phagosomes. Phagosome ...

Immunology and Infection

High Throughput Fluorometric Technique for Assessment of Macrophage Phagocytosis and Actin Polymerization

The goal of fluorometric analysis is to serve as an efficient, cost effective, high throughput method of analyzing phagocytosis and other cellular processes. This technique can be used on a variety of cell types, both adherent and non-adherent, to examine a variety of cellular properties. When studying phagocytosis, fluorometric technique utilizes phagocytic cell types such as macrophages, and fluorescently labeled opsonized particles whose fluorescence can be extinguished in the presence of trypan blue. Following plating of adherent macrophages in 96-well plates, fluorescent particles (green or red) are administered and cells are allowed to phagocytose for varied amounts of time. Following internalization of fluorescent particles, cells are washed with trypan blue, which facilitates extinction of fluorescent signal from bacteria which are not internalized, or are merely adhering to the cell surface. Following the trypan wash, cells are washed with PBS, fixed, and stained with DAPI (nuclear blue fluorescent label), which serves to label nuclei of cells. By a simple fluorometric quantification through plate reading of nuclear (blue) or particle (red/green) fluorescence we can examine the ratio of relative fluorescence units of green:blue and determine a phagocytic index indicative of amount of fluorescent bacteria internalized per cell. The duration of assay using a 96-well method and multichannel pipettes for washing, from end of phagocytosis to end of data acquisition, is less than 45 min. Flow cytometry could be used in a similar manner but the advantage of fluorometry is its high throughput, rapid method of assessment with minimal manipulation of samples and quick quantification of fluorescent intensity per cell. Similar strategies can be applied to non adherent cells, live labeled bacteria, actin polymerization, and essentially any process utilizing fluorescence. Therefore, fluorometry is a promising method for its low cost, high throughput capabilities in the study of cellular processes.

Here we present a protocol to quantify phagocytosis of fluorescent particles by adherent macrophage cell line using a fluorometric method. This method facilitates a high throughput quantification of particle internalization as well as the resulting actin polymerization.

The goal of fluorometric analysis is to serve as an efficient, cost effective, high throughput method of analyzing phagocytosis and other cellular ...

Time-lapse 3D Imaging of Phagocytosis by Mouse Macrophages

Phagocytosis plays a key role in host defense, as well as in tissue development and maintenance, and involves rapid, receptor-mediated rearrangements of the actin cytoskeleton to capture, envelop and engulf large particles. Although phagocytic receptors, downstream signaling pathways, and effectors, such as Rho GTPases, have been identified, the dynamic cytoskeletal remodeling of specific receptor-mediated phagocytic events remain unclear. Four decades ago, two distinct mechanisms of phagocytosis, exemplified by Fc&#947; receptor (Fc&#947;R)- and complement receptor (CR)-mediated phagocytosis, were identified using scanning electron microscopy. Binding of immunoglobulin G (IgG)-opsonized particles to Fc&#947;Rs triggers the protrusion of thin membrane extensions, which initially form a so-called phagocytic cup around the particle before it becomes completely enclosed and retracted into the cell. In contrast, complement opsonized particles appear to sink into the phagocyte following binding to complement receptors. These two modes of phagocytosis, phagocytic cup formation and sinking in, have become well established in the literature. However, the distinctions between the two modes have become blurred by reports that complement receptor-mediated phagocytosis may induce various membrane protrusions. With the availability of high resolution imaging techniques, phagocytosis assays are required that allow real-time 3D (three dimensional) visualization of how specific phagocytic receptors mediate the uptake of individual particles. More commonly used approaches for the study of phagocytosis, such as end-point assays, miss the opportunity to understand what is happening at the interface of particles and phagocytes. Here we describe phagocytic assays, using time-lapse spinning disk confocal microscopy, that allow 3D imaging of single phagocytic events. In addition, we describe assays to unambiguously image Fc&#947; receptor- or complement receptor-mediated phagocytosis.

Here we describe methods using spinning disk confocal microscopy to image single phagocytic events by mouse resident peritoneal macrophages. The protocols can be extended to other phagocytic cells.

Phagocytosis plays a key role in host defense, as well as in tissue development and maintenance, and involves rapid, receptor-mediated rearrangements of ...

A Quantitative Fluorescence Microscopy-based Single Liposome Assay for Detecting the Compositional Inhomogeneity Between Individual Liposomes

Most research employing liposomes as membrane model systems or drug delivery carriers relies on bulk read-out techniques and thus intrinsically assumes all liposomes of the ensemble to be identical. However, new experimental platforms able to observe liposomes at the single-particle level have made it possible to perform highly sophisticated and quantitative studies on protein-membrane interactions or drug carrier properties on individual liposomes, thus avoiding errors from ensemble averaging. Here we present a protocol for preparing, detecting, and analyzing single liposomes using a fluorescence-based microscopy assay, facilitating such single-particle measurements. The setup allows for imaging individual liposomes in a massive parallel manner and is employed to reveal intra-sample size and compositional inhomogeneities. Additionally, the protocol describes the advantages of studying liposomes at the single liposome level, the limitations of the assay, and the important features to be considered when modifying it to study other research questions.

This protocol describes the fabrication of liposomes and how these can be immobilized on a surface and imaged individually in a massive parallel manner using fluorescence microscopy. This allows for the quantification of the size and compositional inhomogeneity between single liposomes of the population.

Most research employing liposomes as membrane model systems or drug delivery carriers relies on bulk read-out techniques and thus intrinsically assumes ...

Bioengineering

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 ...

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a wide variety of disorders including metabolic diseases, premature aging syndromes, and neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson's disease (PD). Maintenance of mitochondrial health depends on mitochondrial biogenesis and the efficient clearance of dysfunctional mitochondria through mitophagy. Experimental methods to accurately detect autophagy/mitophagy, especially in animal models, have been challenging to develop. Recent progress towards the understanding of the molecular mechanisms of mitophagy has enabled the development of novel mitophagy detection techniques. Here, we introduce several versatile techniques to monitor mitophagy in human cells, Caenorhabditis elegans (e.g., Rosella and DCT-1/ LGG-1 strains), and mice (mt-Keima). A combination of these mitophagy detection techniques, including cross-species evaluation, will improve the accuracy of mitophagy measurements and lead to a better understanding of the role of mitophagy in health and disease.

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

Mitophagy, the process of clearing damaged mitochondria, is necessary for mitochondrial homeostasis and health maintenance. This article presents some of the latest mitophagy detection methods in human cells, Caenorhabditis elegans, and mice.

Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a ...

Medicine

Quantitative Analysis of Autophagy using Advanced 3D Fluorescence Microscopy

Prostate cancer is the leading form of malignancies among men in the U.S. While surgery carries a significant risk of impotence and incontinence, traditional chemotherapeutic approaches have been largely unsuccessful. Hormone therapy is effective at early stage, but often fails with the eventual development of hormone-refractory tumors. We have been interested in developing therapeutics targeting specific metabolic deficiency of tumor cells. We recently showed that prostate tumor cells specifically lack an enzyme (argininosuccinate synthase, or ASS) involved in the synthesis of the amino acid arginine1. This condition causes the tumor cells to become dependent on exogenous arginine, and they undergo metabolic stress when free arginine is depleted by arginine deiminase (ADI)1,10. Indeed, we have shown that human prostate cancer cells CWR22Rv1 are effectively killed by ADI with caspase-independent apoptosis and aggressive autophagy (or macroautophagy)1,2,3. Autophagy is an evolutionarily-conserved process that allows cells to metabolize unwanted proteins by lysosomal breakdown during nutritional starvation4,5. Although the essential components of this pathway are well-characterized6,7,8,9, many aspects of the molecular mechanism are still unclear - in particular, what is the role of autophagy in the death-response of prostate cancer cells after ADI treatment? In order to address this question, we required an experimental method to measure the level and extent of autophagic response in cells - and since there are no known molecular markers that can accurately track this process, we chose to develop an imaging-based approach, using quantitative 3D fluorescence microscopy11,12.
Using CWR22Rv1 cells specifically-labeled with fluorescent probes for autophagosomes and lysosomes, we show that 3D image stacks acquired with either widefield deconvolution microscopy (and later, with super-resolution, structured-illumination microscopy) can clearly capture the early stages of autophagy induction. With commercially available digital image analysis applications, we can readily obtain statistical information about autophagosome and lysosome number, size, distribution, and degree of colocalization from any imaged cell. This information allows us to precisely track the progress of autophagy in living cells and enables our continued investigation into the role of autophagy in cancer chemotherapy.

Autophagy is a ubiquitous process that enables cells to degrade and recycle proteins and organelles. We apply advanced fluorescence microscopy to visualize and quantify the small, but essential, physical changes associated with the induction of autophagy, including the formation and distribution of autophagosomes and lysosomes, and their fusion into autolysosomes.

Prostate cancer is the leading form of malignancies among men in the U.S. While surgery carries a significant risk of impotence and incontinence, ...

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.

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 ...

In vitro Quantitative Imaging Assay for Phagocytosis of Dead Neuroblastoma Cells by iPSC-Macrophages

Microglia orchestrate neuroimmune responses in several neurodegenerative diseases, including Parkinson&#39;s disease and Alzheimer&#39;s disease. Microglia clear up dead and dying neurons through the process of efferocytosis, a specialized form of phagocytosis. The phagocytosis function can be disrupted by environmental or genetic risk factors that affect microglia. This paper presents a rapid and simple in vitro&#160;microscopy protocol for studying microglial efferocytosis in an induced pluripotent stem cell (iPSC) model of microglia, using a human neuroblastoma cell line (SH-SY5Y) labeled with a pH-sensitive dye for the phagocytic cargo. The procedure results in a high yield of dead neuroblastoma cells, which display surface phosphatidylserine, recognized as an &#34;eat-me&#34; signal by phagocytes. The 96-well plate assay is suitable for live-cell time-lapse imaging, or the plate can be successfully fixed prior to further processing and quantified by high-content microscopy. Fixed-cell high-content microscopy enables the assay to be scaled up for screening of small molecule inhibitors or assessing the phagocytic function of genetic variant iPSC lines. While this assay was developed to study phagocytosis of whole dead neuroblastoma cells by iPSC-macrophages, the assay can be easily adapted for other cargoes relevant to neurodegenerative diseases, such as synaptosomes and myelin, and other phagocytic cell types.

Neurodegenerative diseases are associated with dysregulated microglia functions. This article outlines an in vitro assay of phagocytosis of neuroblastoma cells by iPSC-macrophages. Quantitative microscopy readouts are described for both live-cell time-lapse imaging and fixed-cell high-content imaging.

Microglia orchestrate neuroimmune responses in several neurodegenerative diseases, including Parkinson&#39;s disease and Alzheimer&#39;s disease. ...

Neuroscience

Quantitative Assessment of Macropinocytosis in mTORC1-Hyperactive Cells using Flow Cytometry

Macropinocytosis is a highly conserved, actin-dependent endocytic process that allows the uptake of extracellular material, including proteins and lipids. In proliferating cells, macropinocytosis can deliver extracellular nutrients to the lysosome, processed into critical macromolecule building blocks. Recent studies have highlighted the dependence of multiple cancers on macropinocytosis, including breast, colorectal and pancreatic cancer. Ras mutations are thought to be the driver events behind macropinocytosis initiation, leading to the activation of cellular anabolic processes via the mTORC1 signaling pathway. Interestingly, mTORC1 can also be activated by macropinocytosis independently of Ras. Therefore, macropinocytosis represents a metabolic vulnerability that can be leveraged to target macropinocytic tumors by limiting their access to nutrients therapeutically.
In Tuberous Sclerosis Complex (TSC) and Lymphangioleiomyomatosis (LAM), mTORC1-hyperactivation leads to enhanced macropinocytosis and metabolic reprogramming. Here, we describe a flow cytometry-based protocol to assess macropinocytosis in mammalian cells quantitatively. TSC2-deficient MEFs are employed, which exhibit aberrant activation of mTORC1 and have been shown to have increased macropinocytosis compared to TSC2-expressing cells. Cells treated with pharmacologic inhibitors of macropinocytosis are incubated with fluorescently labeled, lysine-fixable, 70 kDa dextran, or fluorescently labeled bovine serum albumin (BSA) assayed by flow cytometry. To date, robust image-based techniques have been developed to quantitatively assess macropinocytosis in tumor cells in vitro and in vivo. This analysis provides a quantitative assessment of macropinocytosis in multiple experimental conditions and complements existing image-based techniques.

This protocol provides experimental tools to evaluate macropinocytic uptake of nutrients (carbohydrate and protein) by mTORC1-hyperactive cells. Detailed steps to quantify the uptake of fluorescently labeled dextran and bovine serum albumin (BSA) are described.

Macropinocytosis is a highly conserved, actin-dependent endocytic process that allows the uptake of extracellular material, including proteins and lipids. ...

Automated Imaging and Analysis for the Quantification of Fluorescently Labeled Macropinosomes

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Study of Phagolysosome Biogenesis in Live Macrophages

High Throughput Fluorometric Technique for Assessment of Macrophage Phagocytosis and Actin Polymerization

Time-lapse 3D Imaging of Phagocytosis by Mouse Macrophages

A Quantitative Fluorescence Microscopy-based Single Liposome Assay for Detecting the Compositional Inhomogeneity Between Individual Liposomes

Quantification of Efferocytosis by Single-cell Fluorescence Microscopy

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

Quantitative Analysis of Autophagy using Advanced 3D Fluorescence Microscopy

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

In vitro Quantitative Imaging Assay for Phagocytosis of Dead Neuroblastoma Cells by iPSC-Macrophages

Quantitative Assessment of Macropinocytosis in mTORC1-Hyperactive Cells using Flow Cytometry