Samples of SVF cells from the mammary glands (subcutaneous adipose tissue) were first incubated on ice for 20 minutes in 200 uL of 2% FBS/PBS containing anti-mouse CD16/CD32 Fc Block (clone 2.4G2, BD) (1:200). Cells were then incubated with primary antibody (anti-CD31, 1:200, anti-CD45, 1:200, anti-CD140a, 1:100, all from BioLegend) and were incubated rotating at 4°C for 30 minutes. They were then washed three times with 2% FBS/PBS, and either analyzed using a FACSCantoIITM flow cytometer or sorted by a FACSAriaTM flow cytometer (UT Southwestern Medical Center Flow Cytometry Core Facility). For sorting, cells were initially selected by size, on the basis of forward scatter (FSC) and side scatter (SSC). Live cells were then gated on both SSC and FSC Width singlets, ensuring that individual cells were analyzed. SVF cells isolated from wild-type mice, along with fluorescent-minus-one (FMO) controls, were used to determine background fluorescence levels. Cells were sorted into FBS and then cultured in a 24-well dish. All flow cytometry antibodies were rat-derived and conjugated to APC or PE.
Anti mouse cd16 cd32 fc block clone 2.4g2
Manufactured by BD
Anti-mouse CD16/CD32 Fc Block (clone 2.4G2) is a laboratory reagent used to block Fc receptors on mouse cells. It is commonly used in flow cytometry and other immunological applications to reduce non-specific binding of antibodies.
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Lab products found in correlation
2 protocols using anti mouse cd16 cd32 fc block clone 2.4g2
1
Isolation and Analysis of Adipose-Derived SVF Cells
2
Preparing Splenocyte and Bone Marrow Cells
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To obtain splenocyte and bone marrow cell suspensions, spleens, femurs, and tibias were harvested and single cell suspensions were prepared by homogenizing spleens and flushing each bone with 5 mls of DMEM. For spleen samples red blood cell lysis (144 mM NH4Cl and 17 mM Tris, pH 7.2) was carried out for 5 minutes at 37°C prior to staining. Harvested cells were then suspended in blocking buffer (PBS, 5% bovine serum albumin, 10% normal rat serum, and purified anti-mouse CD16/CD32 [Fc block; clone 2.4G2; BD Biosciences]) for 30 minutes on ice prior to staining with antibodies.
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