Flow cytometry of “Epicardial adipose tissue derived-extracellular vesicles"

The aim of flow cytometry (A60-Micro, Apogee Flow Systems, Hemel Hempstead, UK) experiments was to measure the concentrations of extracellular vesicles (EVs) released from epicardial and subcutaneous adipose tissue (EAT, SAT) in conditioned culture medium from adipose tissue samples collected from 7 patients to investigate whether EAT and SAT secretome (EATs, SATs) show different concentrations of EVs. We hypothesized that EAT secretes a larger number of EVs than SAT. All samples were measured using an autosampler, which facilitates subsequent measurements of samples in a 96-well plate. For this study, samples were measured on two different days (03/01/2021 for 3 patient samples and 04/05/2022 for 4 patient samples). Each day a buffer-only control, antibody in buffer controls, and isotype controls corresponding to the labels in the well plate were measured. Flow rate and scatter calibrations were performed daily. Fluorescence calibration was performed twice during this time period. To automatically determine optimal sample dilutions, apply calibrations, determine and apply gates, generate reports with scatter plots and generate data summaries, we used custom-built software (MATLAB R2020b, Mathworks, Natick, MA, USA).