Adipose tissue SVF isolation and flow cytometry were performed as previously described (46, 47). Briefly, SVF was incubated in Fc Block (rat anti-mouse CD16/32; eBioscience) before staining with anti-CD45 (30-F11), CD3e (145-2C11), CD4 (GK1.5), CD8a (53-6.7), CD11c (N418), and CD64 (X54-5/7.1) (BD Pharmingen). For analysis of Tregs, the samples were fixed after surface staining, permeabilized with a FOXP3 Fix/Perm Buffer Set (421403; BioLegend), and stained with anti-FoxP3 (150D; BioLegend) antibody according to the manufacturer’s protocol. For ILC2 cells, SVF was stained with anti-CD45 (30-F11), B220 (RA3-6B2), CD11b (M1/70), CD11c (N418), Gr-1 (RB6-8C5), CD3e (145-2C11), Thy1.2 (53-2.1) (BioLegend), ST2 (RMST2-2), and Gata3 (TWAJ) (eBioscience), as previously described (11). Samples were analyzed using a BD LSR cell analyzer at the Vision Research Core Facility at the University of Michigan Medical School. Data were analyzed using the FACSDiva v6.2 software (BD Biosciences) and Flowjo ( Flow cytometry gating strategies for Tregs, B cells, macrophages, neutrophils, CD8+ T cells, and ILC2s are shown in fig. S5.

Note: The content above has been extracted from a research article, so it may not display correctly.

Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.

We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.