Anti γδ tcr pe

Cryopreserved cells were thawed, washed, and permeabilized with Perm/Wash solution (BD Biosciences). Cells were then incubated at 4°C for 1 h with fluorescence-conjugated antibodies directed against surface antigens and intracellular cytokines. For detection of the transcription factor FOXP3, a nuclear permeabilization buffer (eBioscience, San Diego, CA, USA) was used in place of the Perm/Wash solution as per manufacturer’s protocol. The following fluorescence-conjugated antibodies were used: anti-CD3 PacBlue, anti-γδ TCR PE, anti-CD27 PECy7, anti-CD25 APC, anti-IFNγ Alexafluor 700, anti-IL17A Alexafluor 647, anti-Ki67FITC (BD biosciences, San Jose, CA, USA); anti-CD45RA QDot655, anti-CD8 QDot605, anti-CD4 QDot605 (Invitrogen, Eugene, OR, USA); and anti-CD39 FITC, anti-IL22 PerCP-Efluor710, and anti-FOXP3 PE (eBioscience, SanDiego, CA, USA).
The entire sample was acquired on a BD LSRFortessa Flow Cytometer using FACSDiva software (BD Biosciences, San Jose, CA, USA). Compensation was performed using compensation beads.

After isolated cardiac cells were treated with mouse Fc Block^TM (BD), samples were stained with anti-CD45.2-Alexa Fluor 488 (BioLegend), anti-CD45.2-APC (BioLegend), anti-Ly6G-PE-Cy7 (BioLegend), anti-CD11b-PErCP-Cy5.5 (BioLegend), anti-CD11b-PE (BD), anti-PDGFRα-APC (BioLegend), anti-CD3ε-PE (BD), anti-CD3ε-PE-Cy5 (TONBO Biosciences), anti-CD4-APC-Cy7 (TONBO Biosciences), anti-CD90.2-APC (eBiosceince), anti-γδTCR-PE (BD), anti-CCR6-APC (RD Systems), anti-B220-APC (BioLegend). For IL-17A staining, cell surface staining was performed, followed by fixation and permeabilisation with Cyofix/Cytoperm^TM (BD) and staining for anti-IL-17A-PE-Cy7 (eBioscience). Dead cells were labelled with the eFluor 780 viability dye (eBioscience). Stained samples were analysed using flow cytometry (AriaIII; BD).