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

Evidence from laboratory and animal studies provides groundwork for understanding mechanisms and potential before human studies continue.

• Level A · Stronger Clinical Evidence
• Level B · Emerging clinical evidence with positive signals
• Level C · Early human research exploring benefits
• Level D · Scientific groundwork from lab and animal studies
• Emerging · Emerging topic under active research

Abstract (original English)

This study examines leukocytes present in lymphoedema (LE) adipose tissue (AT) by multi-colour confocal microscopy. LE AT, collected by liposuction surgery, was digested with collagenase to separate adipocytes from other tissue cells, comprising blood and lymphatic endothelial cells, fibroblasts, and all vessel- and tissue-resident leukocytes - the stromal vascular fraction (SVF). SVF cells were activated with phorbol 12-myristate 13-acetate (PMA) and ionomycin, adding Brefeldin-A to prevent cytokine secretion during the final 4 hours. Cells were incubated with CD11b-FITC and CD40-APC (M1 MØ)' or CD206-APC (M2 MØ) specific antibodies, fixed, permeabilised, then incubated with either (1) anti-TNF-PE, (2) anti- IL-1β-PE, (3) anti-IL-6-PE, (4) anti-IL-4-PE, (5) anti-TGFβ-PE or (6) isotype-IgG-PE (control), and stained with Hoechst 33342, preserved in permanent mounting media and examined by confocal microscopy. The FITC, PE and APC fluorescence channels were set to achieve minimal cross-channel emission using single-colour controls and voltages set for optimal detection by thresholding on isotype-IgG stained activated cells. Finally, transmission and z-stack images were captured. Cells were analysed as regions of interest (ROI) based on Hoechst-33342 then enumerated as FITC + , FITC + APC + or FITC + APC + PE + using an ImageJ script and exported into Excel. This permitted the exa

Evidence level

Evidence from laboratory and animal studies provides groundwork for understanding mechanisms and potential before human studies continue.

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