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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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.

Abstract

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.

Introduction

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....

Protocol

1. Preparation of materials

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

Representative Results

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.......

Discussion

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.......

Acknowledgements

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.

....

Materials

NameCompanyCatalog NumberComments
0.25% TrypsinCorning25053CI0.1% EDTA in HBSS w/o Calcium, Magnesium and Sodium Bicarbonate
1.5 mL Microcentrifuge tubeFisherbrand05-408-129
10-cm Tissue culture dishGreiner Bio-One664160CELLSTAR
15 mL Centrifuge tubeFisherbrand07-200-886
2 L BeakerFisherbrand02-591-33
24-well Tissue culture plateGreiner Bio-One662160CELLSTAR
25 mL Reagent reservoirGenesee Scientific Corporation28-121
500 mL BeakerFisherbrand02-591-30
6-cm Tissue culture dishGreiner Bio-One628160CELLSTAR
8-Channel aspiration adapterIntegra Biosciences155503
8-Channel aspiration adapter for standard tipsIntegra Biosciences159024
95% EthanolDecon Laboratories Inc4355226
Ammonia-free glass cleanerSparkleFUN20500CT
Black 96-well high-content screening microplatePerkinElmer6055300CellCarrier-96 Ultra
Cotton-tipped applicatorFisherbrand23-400-101
CoverslipsFisherbrand12-545-8012 mm diameter
Cytation 5 Cell Imaging Multi-Mode ReaderBiotekCYT5FW
DAPIMillipore Sigma5.08741
Dextran 70 kDa - FITCLife TechnologiesD1822Lysine-fixable
Dextran 70 kDa - TMRLife TechnologiesD1819
DMSOMillipore SigmaD1435
DPBSCorning21031CVWithout Calcium and Magnesium
ForcepsFine Science Tools11251-20Dumont #5
Formaldehyde, 37%Ricca ChemicalRSOF0010-250AACS Reagent Grade
GlycerolFisher BioReagentsBP229-1
Hardening fluorescence mounting mediaAgilent TechS302380-2DAKO
Hoechst 33342Millipore SigmaB2261
Hydrochloric acid (HCl)Fisher ChemicalA144-212Certified ACS Plus, 36.5%–38.0%
Lint-free wipesKimberly-Clark34155Kimwipes
Miscroscope slidesFisherbrand12-544-1Premium plain glass
Multichannel pipetteGilsonFA100138 channels, 0.5–10 µL
Multichannel pipetteGilsonFA10012 12 channels, 20–200 µL
Multichannel pipetteGilsonFA100118 channels, 20–200 µL
Parafilm MPechineyPM996
Plastic wrapKirkland Signature208733Stretch-Tite
Silicone isolatorsGrace Bio Labs Inc66410713 mm Diameter X 0.8 mm Depth ID, 25 mm X 25 mm
Slide adapterBiotek1220548
Wash bottleFisherbrandFB0340922C

References

  1. Lin, X. P., Mintern, J. D., Gleeson, P. A. Macropinocytosis in different cell types: similarities and differences. Membranes. 10 (8), 21 (2020).
  2. Recouvreux, M. V., Commisso, C. Macropinocytosis: a metabolic adapt....

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