Pe cy7 conjugated anti cd69

Cells were stained for surface proteins according to standard protocols. Briefly, cells were washed once with washing buffer (PBS with 1% FBS), then incubated for 30 min at 4°C in a diluted solution of the desired antibodies. Afterwards the cells were washed twice and analyzed on a MACSQuant X or Attune NxT flow cytometer. The antibodies used in this study were the AlexaFluor647-conjugated anti-HA tag antibody (HA.11 from BioLegend), and anti-His6 antibodies (Invitrogen) followed by FITC-coupled anti-mouse IgG (SouthernBiotech) and PE-Cy7-conjugated anti-CD69 (BioLegend). Data were analysed with the FlowJo software.

The following anti-mouse antibodies were obtained from BD PharMingen: PerCP-conjugated anti-CD3e (553067, 1 : 100), PE-conjugated anti-CD8 (557654, 1 : 100), PE-Cy7-conjugated anti-CD8 (552877, 1 : 100), APC-Cy7-conjugated anti-CD8 (557654, 1 : 100), PE-conjugated anti-CD90.1 (561404, 1 : 100), APC-conjugated anti-CD90.1 (557266, 1 : 100), PE-Cy7-conjugated anti-CD62L (560516, 1 : 100), APC-Cy7-conjugated CD62L (560514, 1 : 100), and APC-conjugated anti-IFN-γ (554413, 1 : 100). Biolegend: PE-conjugated anti-CD44 (103008, 1 : 100), PE-Cy7-conjugated anti-CD44 (103030, 1 : 100), PE-Cy7-conjugated anti-CD69 (104512, 1 : 100), and APC-conjugated anti-CD103 (121414, 1 : 100). Flow cytometry data were acquired with a FACS Canto II flow cytometer (BD Biosciences) and data were analyzed with Flowjo software (Tree Star).