A subscription to JoVE is required to view this content. Sign in or start your free trial.
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, 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 µm. These puncta can be microscopically imaged and subsequently quantified to determine the extent of macropinocytosis in the cell - 'the macropinocytic index'.
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.
Figure 1: Schematic of the automated assay to determine the 'macropinocytic index' in adherent cells. Created using BioRender. Please click here to view a larger version of this figure.
1. Preparation of materials
2. Preparation of cells
3. Macropinosome labeling
Figure 2: Placing coverslips on a microscope slide with silicone isolators. (A) Silicone isolators are pressed and adhered to a microscope slide. (B) The entire microscope slide can be populated with a total of 3 isolators, resulting in even spacing and reproducible localization of the coverslips. (C) For each coverslip, add a drop of fluorescence mounting media on the microscope slide within the open space of the isolator. (D) Using forceps, pick up a coverslip from the 24-well plate and place it upside down on the drop of mounting media. (E) When bubbles are present between the coverslip and microscope slide, gently tap the coverslip using closed forceps to remove bubbles. Created using BioRender. Please click here to view a larger version of this figure.
Figure 3: Rinsing the 96-well microplate to prepare for fixation. (A) Empty the microplate of media into a 5 L beaker by manually flicking. (B) Vertically and at a slight angle, slowly submerge the microplate in a 2 L beaker filled with ice-cold PBS. (C) Empty the microplate of PBS into the 5 L beaker by manually flicking. Repeat the wash steps as described in B two times. (D) After emptying the PBS in the microplate for the last time, add 100 µL 3.7% formaldehyde to the wells, using a multichannel pipette. Created using BioRender. Please click here to view a larger version of this figure.
4. Automated macropinosome imaging
Images of macropinosomes can be captured using a standard fluorescent microscope, as previously described19,20. However, such a procedure can be improved upon in terms of efficiency through automation, especially when assessing numerous different cell culture conditions. Automation of image acquisition can be accomplished via a cell imaging multi-mode plate reader, which decreases effort by reducing handling procedures and, importantly, increases data reproducibility and reliability by acquiring images in an unbiased fashion. Multiple imaging systems are commercially available, and directions will differ between instruments. Here, acquiring images using a Cytation 5 is described. However, the protocol below can be tailored to each individual instrument by adhering to the following guidelines:
Figure 4: Optimization of conditions for image acquisition. (A) Increasing the glycerol concentration increases TMR-dextran fluorescence, as determined in AsPC-1 cells treated with EGF. (B) Example coordinates of imaging beacons for automatic image acquisition when using the 24-well plate with coverslips format. The bar graph shows the average relative fluorescence with SEM of 5 experiments. Statistical significance was determined by two-way ANOVA, relative to PBS. ** p < 0.01; *** p < 0.001. Please click here to view a larger version of this figure.
Figure 5: Control conditions for assessing macropinocytosis in PDAC cells. (A) AsPC-1 cells display macropinocytosis in response to 100 ng/mL EGF stimulation for 5 min or glutamine deprivation for 24 h. For image acquisition, picture frames of 4 x 4, 3 x 3, 2 x 3, or 2 x 2 were taken to determine the influence of the number of photos on data quality. (B) MIA PaCa-2 cells show constitutive macropinocytosis that is inhibited by 30-min treatment with 75 µM EIPA or 2-h treatment with 10 µM EHop-016. Picture frames were taken as in A. Scale bar = 25 µm. Bar graphs show the average relative macropinocytic index with SD of 1 experiment with 4 replicates. Statistical significance was determined by two-way ANOVA relative to the +Q or vehicle condition. *** p < 0.001 Please click here to view a larger version of this figure.
5. Determining the macropinocytic index
The 'macropinocytic index' is the extent of cellular macropinocytosis that is determined by quantifying fluorescent dextran uptake per cell using microscopic imaging19. To this end, the acquired images are used to determine the amount of internalized dextran by measuring the total fluorescence intensity or fluorescence-positive area and the total number of cells as determined by DAPI staining. This analysis can be performed with open-source image processing and analysis software, such as Cell Profiler or FIJI/ImageJ, as previously described19,20. However, when working with a multi-mode plate reader the software provided with the instrument may include built-in analysis applications that can be used for the purposes of computing the macropinocytic index. In some cases, the built-in software analysis pipeline may not be completely apparent to the user. It is therefore recommended to validate the software at an early stage by comparison with a non-automated procedure, such as Cell Profiler or FIJI/ImageJ. This protocol can be adapted to other image processing and analysis software tools by adhering to the following general instructions:
6. Addition of treatments
Cell treatments (small molecules, biologics, growth factors, metabolites etc.) can be incorporated at any stage of the protocol, and the precise timing will depend on the goals and aims of the study.
Figure 6: Performing a dose-response curve for macropinocytosis inhibitors. Example data obtained when testing known macropinocytosis inhibitors in a new cell line. PATU8998T cells were used for the 96-well microplate format and treated for 2 h and 30 min with the indicated concentrations of (A) EHop-16 and (B) EIPA, respectively. Comparison of results obtained through image analysis by the Gen5 software or ImageJ shows no significant differences between the two approaches as indicated by ns in (A). Scale bar = 25 µm. Bar graphs show the average and SD of a single experiment with 4 replicates. Statistical significance was determined by one- or two-way ANOVA, compared to untreated conditions. * p < 0.05; *** p < 0.001. Please click here to view a larger version of this figure.
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...
C.C. is an inventor on an issued patent titled ''Cancer diagnostics, therapeutics, and drug discovery associated with macropinocytosis,'' Patent No.: 9,983,194.
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 |
Request permission to reuse the text or figures of this JoVE article
Request PermissionThis article has been published
Video Coming Soon
Copyright © 2025 MyJoVE Corporation. All rights reserved
We use cookies to enhance your experience on our website.
By continuing to use our website or clicking “Continue”, you are agreeing to accept our cookies.