An improved method for high-throughput quantification of autophagy in mammalian cells

Autophagy is a cellular homeostatic pathway with functions ranging from cytoplasmic protein turnover to immune defense. Therapeutic modulation of autophagy has been demonstrated to positively impact the outcome of autophagy-dysregulated diseases such as cancer or microbial infections. However, currently available agents lack specificity, and new candidates for drug development or potential cellular targets need to be identified. Here, we present an improved method to robustly detect changes in autophagy in a high-throughput manner on a single cell level, allowing effective screening. This method quantifies eGFP-LC3B positive vesicles to accurately monitor autophagy. We have significantly streamlined the protocol and optimized it for rapid quantification of large numbers of cells in little time, while retaining accuracy and sensitivity. Z scores up to 0.91 without a loss of sensitivity demonstrate the robustness and aptness of this approach. Three exemplary applications outline the value of our protocols and cell lines: (I) Examining autophagy modulating compounds on four different cell types. (II) Monitoring of autophagy upon infection with e.g. measles or influenza A virus. (III) CRISPR/Cas9 screening for autophagy modulating factors in T cells. In summary, we offer ready-to-use protocols to generate sensitive autophagy reporter cells and quantify autophagy in high-throughput assays

MOESM1 of Increased susceptibility of CD4+ T cells from elderly individuals to HIV-1 infection and apoptosis is associated with reduced CD4 and enhanced CXCR4 and FAS surface expression levels

Additional file 1: Figure S1. Increased levels of apoptosis in lymphocytes from elderly donors. Gating strategy of flow cytometry analysis of death and apoptosis rates from mock or HIV-1 NL4-3 reporter virus infected GFP+ or GFP− cells. Figure S2. Expression of activation markers and viral LTR activity in HIV-1 infected PBMC cultures from young and elderly individuals. (A, B) Representative primary data and statistical evaluations of the expression levels of (A) CD69 and (B) CD25 on X4 or R5 HIV-1 NL4-3 reporter virus infected GFP+ or GFP− cells from young (Y) and elderly (O) blood donors. (C) GFP expression levels in PBMC cultures from young and elderly donors infected with X4 or R5 HIV-1 NL4-3 reporter constructs. Each symbol represents the result obtained for one individual PBMC donor from the young (blue) or elderly (red) groups