Multi-parameter Analysis of Germinal Center B Cells

Inguinal dLNs and spleens were prepared as described42 (link). Cells were transferred to 96 well V-bottom plates (Thermo Scientific, UK) and centrifuged (300 × g, 5 min). Supernatants were then discarded and cells were resuspended with addition of Fc Receptor Block (1:50) (eBioscience, UK) for 15 min. Following a wash in FACS buffer, cells were stained with anti-GL7 AlexaFluor 647 (eBioscience, UK) at 1:100 dilution, anti-CD95 PE (eBioscience, UK) at 1:100 dilution and anti-B220 PeCy7 (eBioscience, UK) at 1:200 for 30 min at RT in the dark. After this incubation, cells were washed twice and resuspended in FACS buffer and data were acquired on a LSRII (BD Bioscience, UK) and analyzed by FlowJo (TreeStar Inc, USA).