Anti cd16 cd32 fc blocking antibody clone 2.4g2

The following anti-mouse antibodies were used: anti-CD16/CD32 Fc blocking antibody (clone 2.4G2) and anti–CD45-PE (clone 30-F11) (BD Biosciences); anti–CD24-FITC (clone M1/69), anti–SCA1-APC (clone D7) anti–Gr1-eFlour450 (clone RB6-8C5), anti–Ly6G-APC (clone 1A8-Ly6g), anti-F4/80 PerCP/Cy5 (clone BM8), anti–CD11b-PE-Cy7 (clone M1/70), anti–CD11c-APC-eFluor780 (clone N418), anti–CD4-FITC (clone 6K1.5), anti–CD8-PE (clone 53-6.7), and anti–B220-APC (clone RA3-6B2) (eBioscience); anti–CD61-Alexa Fluor 647 (clone 2C9.62[HMβ3-1]), anti–Ly6C FITC (clone HK1.4), anti–CD49b-eFluor450 (clone DX5), annexin V–APC (clone B217656) (BioLegend); and propidium iodide–PerCP (clone V13245) (Life Technologies).

Spleens, inguinal lymph nodes, blood and peritoneal fluids were isolated from WT T505A mice. For blood samples, red blood cell lysis was performed using Pharm Lyse lysis buffer (BD) according to manufacturer's instructions. Following the generation of single-cell suspensions, total cell counts were calculated in the presence of trypan blue (Sigma–Aldrich) using a haemocytometer, followed by incubation with anti-CD16/CD32 Fc-Blocking antibody (clone 2.4G2 (BD Pharmingen)). Distinction between live and apoptotic/necrotic cells was performed based on staining with LIVE/DEAD Aqua (Life Technologies). For cell surface marker detection cells were stained with a combination of FITC, PE, APC, PerCP-Cy5.5, APC-Cy7 conjugated monoclonal antibodies (BD Pharmigen). For detection of Foxp3, intracellular staining was performed using a Fix/Perm kit (eBiosciences). See below for additional antibody information. All samples were acquired using a three laser FACS Canto II (BD Bioscience) and the data were subsequently analysed using FlowJo (version 9 or X; Treestar, U.S.A.).
Antibodies used in flow cytometric analysis of lymphoid tissue cells