Pe anti bcl 6

Freshly isolated cells were stained on the same day as the isolation. For staining, cells were incubated with LIVE/DEAD™ Fixable Green Dead Cell Stain Kit (Fisher Scientific) and surface protein antibodies at 4 °C for 30 min. Stained cells were washed with FACS buffer and resuspended in Foxp3 Fixation/Permeabilization buffer (eBiosciences) according to manufacturer’s instructions. Fixed cells were stained for Bcl6 with PE anti-Bcl-6 (BD Biosciences) at room temperature for 30 mins. Cells were washed twice with 2 mL 1x permeabilization buffer before being resuspended in FACS buffer. Flow cytometric data were collected using a BD LSR Fortessa analyzer (BD Biosciences) with FACS Diva version 8.01 software. Data was subsequently analysed with FlowJo (version 10, Tree Star). Single leukocytes were identified based on forward and side scatter parameters, and expression of CD45. Dead and lineage positive cells were excluded using LIVE/DEAD™ Fixable Green Dead Cell Stain Kit (Fisher Scientific) and lineage markers (CD1a, CD14, CD34, CD94, BDCA2, FcεRIα, CD123, TCRγδ, CD19). T helped cells were gated at CD45⁺CD3⁺CD4⁺ before further gating as CXCR5⁺PD1⁺ TfH cells. ILCs were gated as Lin^-CD3^-CD127⁺ before further gating into CD117^-CRTH2^- ILC1s and CD117⁺CRTH2^- ILC3s, ILC3s were then gated as NKp44⁺NRP1⁺ or NKp44^-NRP1^-.