5 laser lsr 2 flow cytometers

Fluorochrome-coupled anti-mouse CD4 (GK1.5), CD8α (53–6.7), CD45RB (C363.16A), granzyme B (NGZB), IFNγ (XMG1.2), IL-17a (TC11–18H10), Ki67 (solA15), TCRβ (H57–597) and ghost viability dyes were purchased from ThermoFisher, BD Bioscences or Tonbo. Biotinylated anti-CD8α (53–1.7) and CD19 (1D3) antibodies were purchased from Tonbo. The Invitrogen Vybrant CFDA SE cell tracer kit was purchased from ThermoFisher Scientific. Anti-CD40 antibody was purchased from BioXcell. Surface cell staining was performed following conventional techniques. For intracellular cytokine staining, cells were stimulated with PMA and ionomycin in the presence of Golgi Stop (BD Biosciences) for 4hr prior to staining. Extracellular markers were stained, cells were fixed briefly with 2% paraformaldehyde, followed by permeabilization and intracellular staining. For intracellular cytokine and granzyme B staining, the BD Cytofix/Cytoperm kit was used according to the manufacturer’s instructions. For intracellular Ki67 staining, the eBioscience transcription factor staining buffer set was used according to the manufacturer’s instructions. All stained samples were acquired using BD FACS Canto II, 3-or 4-Laser Fortessa, or 5-Laser LSR II flow cytometers (BD Bioscences). Data were analyzed using FlowJo software (Tree Star).