Anti cd27 apc efluor780

A total of 1 x 10⁶ PBMCs were stained with LIVE/Dead fixable violet stain (Invitrogen) for 15 minutes at room temperature (RT), followed by a wash step with phosphate-buffered saline (PBS). Cells were then re-suspended in 50 μL of human serum and incubated with PE-conjugated HLA class II tetramer for 1 h at 37°C and 5% CO₂. After three washes with staining buffer (1xPBS supplemented with 0.5% BSA and 2 mM EDTA), the cells were co-stained for 15 minutes at 4°C with saturating amounts of different combinations of the following antibodies: anti-CD3 AmCyan (SK7, BD Biosciences), anti-CD4 PerCPCy5.5 (RPA-T4, eBioscience), anti-CD4 PE-CF594 (RPA-T4, BD Biosciences), anti-CD4 APC-Cy7 (RPA-T4, Biolegend), anti-CD14 Pacific Blue (HCD14, Biolegend), anti-CD19 eFluor450 (H1B19, eBioscience), anti-CD25 APC-Cy7 (BC96, Biolegend), anti-CD27 APC-eFluor780 (O323, eBioscience), anti-CD28 PE-Cy7 (28.2, Biolegend), anti-CD45RA AF700 (H1 100, Biolegend), anti-CD57 APC (HCD56, Biolegend), anti-CD69 PE-Cy7 (FN50, Biolegend), anti-CD127 PerCP-Cy5.5 (HIL-7R-M21, BD), anti-CCR7 FITC (150503, R&D), anti-CX3CR1 PerCP-Cy5.5 (2A9-1, Biolegend), anti-FasL AF488 (14C2, AbD Serotec), anti-NKG2D PE-CF594 (1D11, BD Biosciences), anti-PD-1 PerCP-Cy5.5 (EH12.2H7, Biolegend) and anti-Tim3 APC (F38-2E2, eBioscience). Following a final wash cells were re-suspended in 200 μL of staining buffer for acquisition.

Unactivated and activated Jurkat cells were seeded 5 × 10⁴ cells/mL in 12 well plates in complete RPMI. Jurkats were activated with plate bound anti-CD3 (Biolegend, CA, USA) and anti-CD28 (Ancell, MN, USA) monoclonal antibodies for 24 h. Cells were treated for 24 or 48 h with 0.1% DMSO vehicle control or 10 µM of pyrazinib (P3). Cells were washed and blocked and then stained with anti-CD62 PerCp-Cy5 (Abcam, UK), anti-CD69 PE (BD Biosciences, CA, USA), anti-CD45RO FITC (Immunotools, Germany), anti-CD45RA V500 (BD Biosciences) and anti-CD27 APC eFluor780 (eBioscience. CA, USA) for 20 min at 4 °C in the dark. Cells were washed and resuspended in 300 μL FACS buffer and acquired on a FACSCanto II flow cytometer (BD Biosciences).