Fig. 2

Immunophenotyping following tissue explant culture and enzymatic digestion. Gating strategy of multicolor flow cytometry. (A) Selection of cells of interest and removal of debris based on side scatter area (SSC-A) and forward scatter area (FSC-A), (B) Singlets cell selection based on SSC-A and side scatter height (SSC-H), (C) Selection of CD90⁺ cells, (D) Selection of CD45⁻ cells, (E) Selection of CD31⁻ cells. (F) Representative dot plot following tissue explant culture (TEC). Pericytes were identified as CD31⁻CD34⁻CD45⁻CD90⁺CD146⁺, transitional pericytes (TP) as CD31⁻CD34⁺CD45⁻CD90⁺CD146⁺, and adventitial stem cells (ASCs) as CD31⁻CD34⁺CD45⁻CD90⁺CD146⁻. (G) Representative dot plot following enzymatic digestion (ED). Subtypes were identified as stated above. (H) Selection of CD271⁺ cells identified as CD31⁻CD45⁻CD90⁺CD271⁺. Gating was performed based on non-stained cells, fluorescence-minus-one (FMO) and mouse IgG1κ isotype controls for each fluorophore. (I) Box plot showing the distribution (%) of immunophenotypes of ASCs, TPs, pericytes, and CD271⁺ stem cells from cryopreserved microfragmented adipose tissue following isolation by TEC and ED, respectively, when analyzed by flow cytometry at passage 4. The data is shown as mean values with standard deviation. ns: non-significant, *: statistically significant p-value < 0.05 when analyzed with paired t-tests