A multi-colour confocal microscopy method for identifying and enumerating macrophage subtypes and adherent cells in the stromal vascular fraction of human adipose

This study examines leukocytes present in lymphoedema (LE) adipose tissue (AT) by multi-colour confocal microscopy. LE AT, collected liposuction surgery, was digested with collagenase to separate adipocytes from other cells, comprising blood and lymphatic endothelial fibroblasts, all vessel- tissue-resident - the stromal vascular fraction (SVF). SVF cells were activated phorbol 12-myristate 13-acetate (PMA) ionomycin, adding Brefeldin-A prevent cytokine secretion during final 4 hours. Cells incubated CD11b-FITC CD40-APC (M1 MØ)‚ or CD206-APC (M2 MØ) specific antibodies, fixed, permeabilised, then either (1) anti-TNF-PE, (2) anti- IL-1?-PE, (3) anti-IL-6-PE, (4) anti-IL-4-PE, (5) anti-TGF?-PE (6) isotype-IgG-PE (control), stained Hoechst 33342, preserved permanent mounting media examined The FITC, PE APC fluorescence channels set achieve minimal cross-channel emission using single-colour controls voltages for optimal detection thresholding on isotype-IgG cells. Finally, transmission z-stack images captured. analysed as regions of interest (ROI) based Hoechst-33342 enumerated FITC+, FITC+APC+ FITC+APC+PE+ an ImageJ script exported into Excel. permitted examination >9000 individually, per sample. method allows analysis a high number heterogeneous defined any subtype combination investigators' choice surface intracellular expression profiles. Fibroblasts, producing can also be cell count data correlated clinical laboratory data.