Three laser facscanto 2

For FACS cell cycle analysis, cells were incubated with 10 μM EdU (Life Technologies) for 15 min in media before trypsinization and fixation in 70% ice cold ethanol. Cells were stored at −20°C before staining. Fixed cells were washed twice in 1× PBS + 0.1% Triton-X 100 before resuspension in Click-It EdU reaction mix (Alexa-555; Thermo Fisher Scientific) for 30 min while rotating. Cells were washed twice in 1× PBS + 1% BSA and incubated in 1 μg/ml DAPI in 1× PBS for 30 min before analysis. Samples were analyzed on either a three-laser FACSCanto II (BD Biosciences) or a four-laser LSR II (BD Biosciences) and data acquired using DIVA software (BD Biosciences). DNA content was analyzed based on DAPI fluorescence (PacBlue-A), and DNA replication was analyzed based on Alexa-555 fluorescence (PE-A). Doublet discrimination was used to remove doublets and clumped cells using DAPI-A/DAPI-W measurements. Data were analyzed using FlowJo v.10 software (BD Biosciences). Cell cycle distributions were determined by gating EdU positive versus negative, as determined by single color control, and by 2N versus 4N DAPI content.

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