Automated Imaging and Analysis for the Quantification of Fluorescently Labeled Macropinosomes

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. Here, we describe 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.

Cell polarity proteins promote macropinocytosis in response to metabolic stress

“…Metabolic stress caused by glutamine deprivation stimulates macropinocytosis; however, a comprehensive understanding of modulators that can control uptake in the context of nutrient stress is lacking 5 . In order to gain insight into the pathways that might broadly function in macropinocytosis in the context of glutamine stress, we performed a high-throughput (HT) siRNA screen using a HT-compatible macropinocytosis assay that we previously developed 13, 14 . For the screen, we used AsPC-1 cells since these cells display robust stimulation of macropinocytosis upon glutamine starvation (Extended Data Fig.…”

“…Calculation method of the macropinocytic index was the same as described above. Both approaches have been previously compared and have shown to provide the same results 14 .…”

“…The macropinocytic index was calculated by the total particle area per cell. Alternatively, images were automatically captured at 40X magnification using the BioTek Cytation 5 (Agilent Technologies) from the Sanford Burnham Prebys Cell Imaging Core and were subjected to automated analysis using the BioTek Gen5 software 14 . Calculation method of the macropinocytic index was the same as described above.…”

Surfactant deficiency increases SARS-CoV-2 canonical and non-canonical viral entry to lung epithelial cells and exacerbates intrapulmonary inflammatory responses

“…Lowly expressed genes were ltered out by applying the following criterion: estimated counts (from RSEM) ≥ number of samples * 5. Filtered estimated read counts from RSEM were compared using the R Bioconductor package DESeq2 v1.22.2 based on generalized linear model and negative binomial distribution [151]. Genes with Benjamini-Hochberg corrected p-value < 0.05 and fold change ≥ 2.0 or ≤ -2.0 were selected as differentially expressed genes.…”

The lung employs an intrinsic surfactant-mediated inflammatory response for viral defense

“…Endosomes (specifically macropinosomes) were labeled and quantitated utilizing previously established procedures (152)(153)(154). Macropinosomes in live cells were labelled by their uptake of high molecular weight fluorescent dextran (FITC-Dextran).…”