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.

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.

The prevalence of “Long COVID”, including among vaccinated patients, is just one of the conundrums that indicate how much remains unknown about the lung’s response to viral infection, particularly to SARS-CoV-2 for which the lung is the point of entry. Therefore, we used an in vitro human lung system to enable a prospective, unbiased, sequential single cell level analysis of pulmonary cell responses following infection by multiple strains of SARS-CoV-2. By starting with human induced pluripotent stem cells (hiPSCs) and emulating lung organogenesis, three-dimensional lung organoids were generated and infected in which several unexpected but pertinent insights emerged. First, SARS-CoV-2 tropism is much broader than previously believed: most lung cell types can be infected, if not through a canonical receptor-mediated route (e.g., via ACE2) then via a non-canonical “backdoor” endocytosis/micropinocytosis route. Such entry can be abrogated by FDA-approved endocytosis blockers, suggesting novel adjunctive therapies. Regardless of route-of-entry, the virus triggers a heretofore unrecognized lung epithelial cell-intrinsic autonomous innate immune response involving interferons and cytokine/chemokine production in the absence of hematopoietic cells or their derivatives. The virus can spread rapidly throughout human lung organoid cell cultures resulting in mitochondrial apoptosis mediated by the pro-survival protein Bcl-xL. This host cytopathic response to the virus may help explain persistent inflammatory signatures in a dysfunctional pulmonary environment of long COVID. The host response to the virus is, in part, dependent on the presence of pulmonary Surfactant Protein-B (SP-B), which plays an unanticipated role in signal transduction, viral resistance, dampens systemic inflammatory cytokine production, and minimizes the induction of apoptosis.