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