Isolation of stromal vascular fraction cell suspensions from mouse and human adipose tissues for downstream applications

Figures

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  Figure 1. Examples of how samples look before and after different steps of the adipose tissue digestion protocol (A) Ensure adipose tissue samples are thoroughly washed and processed to remove residual blood and non-adipose tissue (e.g., connective tissue, cauterized tissue). (B) If there are still lumps of adipose tissue visible within the sample (Left) after step 3 of the digestion protocol, then return samples to the shaker-incubator until they form a homogeneous mixture (Right). (C) If samples look partially digested (Left) after step 5, with adipose tissue still visible, then carry out the additional re-digestion step before proceeding to step 6. If samples look completely digested (Right), with only lipid droplets visible, then proceed to step 6 of the adipose tissue digestion protocol.

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  Figure 2. Bead enrichment and flow cytometry gating strategy for isolation of conventional dendritic cell subsets from mouse adipose tissue (A) The beneficial effects of magnetic bead enrichment (dead cell removal and CD11c+ enrichment) on live cells and conventional dendritic cell populations within a sample. (B) An example flow cytometry gating strategy for the isolation of conventional dendritic cell subsets (cDC1 and cDC2). Note that stromal vascular fractions taken from mouse adipose tissue were pooled (to increase yield) and cDCs expressing a Zbtb46-GFP reporter are shown in this example.

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  Figure 3. Example flow cytometry gating strategy for quantifying immune cell populations from digested adipose tissues Lymphocyte populations (A) and myeloid populations (B) analyzed from the stromal vascular fraction of human adipose tissue via multi-color flow cytometry.

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  Figure 4. Samples contaminated with different non-adipose tissues can affect experimental data (A) Tissue samples that sink in Wash/Digestion buffer are most likely not adipose tissue. When acquiring adipose tissue (AT) from mice, if testes (middle) or lymph nodes (LN) (right) are accidentally acquired, ensure they are removed before digestion. (B) Tissue contaminants can distort different cell populations. As shown via flow cytometry analysis, when lymph nodes from a mouse are present during digestion, samples have increased CD45+ cells. This can compromise studies investigating T cell (CD45+ CD3+) abundance (right).