Cd21 percp cy5

Cells were stained using Abs specific for mouse Ags: CD45R (B220) (various fluorochromes) (BD Pharmingen, BioLegend, San Diego, CA and Tonbo Biosciences); IgM (various fluorochromes, Jackson ImmuoResearch Laboratories, West Grove, PA); CD19 eFlour450, CD25 APC, MHC II APC (Tonbo Biosciences); CD43 PE, BP-1 PE, CD117 PE, CD24 (HSA) PE-Cy7, IgD FITC (BD Pharmingen); CD21 PerCP/Cy5.5 (BioLegend). IC staining was performed with IC fixation and permeabilization buffer (eBiosciences). Methanol fixation was performed for IC phospho (p)-S6R detection. Abs used for IC staining were p-ribosomal S6 protein (S6R) S235/236 PE (eBiosciences); p-AMPKα T172, c-Myc AF488 and p-Ulk1 S555 (Cell Signaling Technology, Danvers, MA). Donkey anti-rabbit Alexa-Fluor 647 (Life Technologies, Carlsbad CA) secondary Ab was used to detect unlabeled primary Abs. Data was collected using FACS Canto II or LSR II flow cytometers (BD Biosciences) and analyses were performed using FlowJo software (TreeStar, Ashland, OR).

Splenocytes were rested in cRPMI for 20 mins at 37°C, before staining. Splenocytes from one genotype (5×10⁶) were labeled with 1μg Cy5 dye in cRPMI (GE healthcare, Chalfont, UK) for 5 mins at room temperature and the other genotype was left unstained. Cy5 labeled cells were washed and mixed with unlabeled cells. These cells were then loaded with INDO-1 AM (Invitrogen) in RPMI media for 30 minutes at 37°C. Following washing the cells were stained with CD19 FITC, CD23 PE and CD21 PerCP Cy5.5 (all Biolegend) and resuspended in HBSS (Gibco) and kept on ice. Cells were then warmed to 37°C prior to running the sample and ran for 30 seconds to establish a baseline, 10μg/ml anti-IgM Fab₂ (Jackson Immunoresearch, WestGrove, PA) was added at 30 seconds to stimulate B cells. At 2 minutes, 10mM CaCl₂ (Sigma Aldrich, St. Louis, MO) was added and the cells run until 5 minutes. At 5 minutes 1μg/ml of ionomycin (EMD Biosciences, La Jolla, CA) was added and the sample run until 7 minutes.