Adipose Stromal Vascular Fraction Isolation

Perigonadal VATs were minced and digested with type II collagenase (Sigma). Stromal vascular fraction (SVF) cells were obtained as described by Anderson et al. (18 (link)). After blocking with anti-CD16/32 antibodies (BD Biosciences, San Jose, CA), cells were stained with fluorophore-conjugated antibodies. Allophycocyanin (APC)-Cy7-B220, APC-TCRβ, phycoerythrin (PE)-Cy7-CD8, PerCP-Cy5.5-CD44, Alexa Fluor 488–CD62L, PE-Cy7-CD45.2, BV421-F4/80, APC-Cy7-CD11c, fluorescein isothiocyanate (FITC)-CD11b, PE–Siglec F, APC-CD11c, Pacific Blue–CD3, Pacific Blue–CD8ɑ, Pacific Blue–CD19, FITC-FCεIɑ, FITC–Pan NK, APC-Cy7-CD25, PerCP-Cy5-CD127, PE-IL33R, APC-CD11b, Alexa Fluor 488–CD45.1, and PerCP-Cy5.5-CD86 antibodies were from BioLegend (San Diego, CA). FITC-CD4 and eFluor 605-CD4 antibodies were from BD Biosciences and eBioscience (San Diego, CA), respectively. DAPI (Invitrogen, Carlsbad, CA) or 7-aminoactinomycin D (BioLegend) was added as viability dye. Flow cytometry experiments were performed on the BD LSR II and analyzed using FlowJo software (Tree Star, Ashland, OR).

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Liu L., Inouye K.E., Allman W.R., Coleman A.S., Siddiqui S., Hotamisligil G.S, & Akkoyunlu M. (2018). TACI-Deficient Macrophages Protect Mice Against Metaflammation and Obesity-Induced Dysregulation of Glucose Homeostasis. Diabetes, 67(8), 1589-1603.

Publication 2018

type II collagenase anti-CD16/32 antibodies APC-Cy7-CD25 APC-CD11b PerCP-Cy5.5-CD86 FITC-CD4 DAPI 7-aminoactinomycin D

Alexa fluor 488 Allophycocyanin Antibodies Blocking antibodies Cd11b Cd25 Cd44 Cd62l Cells Collagenase Cy5 5 Dapi Flow cytometry Fluorescein Isothiocyanate Phycoerythrin Siglec Stromal vascular fraction Tcrβ Tree Vats

Corresponding Organization : United States Food and Drug Administration

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Variable analysis

independent variables

Type II collagenase (Sigma) used for mincing and digesting perigonadal VATs

dependent variables

Composition and characteristics of stromal vascular fraction (SVF) cells

control variables

Anti-CD16/32 antibodies used for blocking
Fluorophore-conjugated antibodies used for staining
DAPI or 7-aminoactinomycin D used as viability dye
Flow cytometry experiments performed on BD LSR II and analyzed using FlowJo software

controls

Not explicitly mentioned
Not explicitly mentioned

Annotations

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