Anti cd8 bv570

The following mouse flow cytometry conjugated antibodies were used at the concentration suggested on the manufacturer’s data sheet for surface or intracellular staining: Live/Dead Ghost UV450, anti-CD45 BUV805, anti-PD1 BV421, anti-CD8 BV570, anti-NKG2D FITC, anti-CD278 BV785, anti-IFNγ PerCP-Cy5.5, anti-PD-L1 PE-Dazzle 594, anti-NK1.1 Pe-CY5, anti-CD3 Pe-Fire700, anti-CD4 AlexaFluor700, anti-Ki-67 Pacific Blue, anti-FoxP3 Alexa532, anti-Granzyme B PE, anti-IL-17A APC all purchased from Biolegend or Thermofisher. For intracellular staining of FoxP3, Granzyme B, IL-17A, and Ki-67, the cells were fixed and permeabilized using cold 70% ethanol. Immuno-stained cell percentages were assessed by a Cytek Aurora flow cytometer and analyzed by Flow-Jo software.

Testes were collected from adult C57BL/6 (JAX®mice, stock no. 000664) mice and enzymatically and mechanically dissociated into a single cell suspension enriched for interstitial cells as described above. The single cell suspension is stained with a series of cell surface markers including anti-CD8-BV570 (1:300; Biolegend 100739), anti-CD3-PE/Cy7 (1:300; Biolegend 100219), anti-CD4-BV510 (1:300; Biolegend 100449), anti-IL7r-PE/Cy5 (1:300; Biolegend 135015). However, for intracellular staining - the ILCII cells were first incubated with PMA (10 ng / mL), ionomycin (10 μM), with the addition of Golgi-stop reagent (BD Bioscience, San Jose, CA) for 4 h at 37°C and subsequently stained for anti-IL4-PE (1:300; Biolegend 504103), anti-IL-13-APC (1:300; Novus 011818), and anti-Gata3-BV421 (1:300; Biolegend 653813) for 30 minutes prior to flow cytometry.