Anti helios pacificblue

For flow cytometric analysis, cells were washed with phosphate buffered saline (PBS) and stained with a fixable viability dye (Zombie NIR Fixable Viability Kit, Biolegend, San Diego, CA, USA), followed by staining of surface markers. Where indicated, intracellular staining of transcription factors was performed by fixing and permeabilizing the cells using buffers and instructions of commercially available kit (FixPerm # AB_2869008, BD, Franklin Lakes, NJ, USA). Antibodies used were supplied from BioLegend (San Diego, CA., USA) (anti-CD25-PE # 101903, anti-CD4-PerCP # 100431, anti-CD4-BrilliantViolet421 # 100437, anti-CD44-APC # 103012, anti-CD62L-PE/Cy7 # 104418, anti-FoxP3-AlexaFluor647 # 320013, and anti-Helios-PacificBlue # 137210). Flow cytometric analysis was performed with BD FACS Verse^TM cytometer and FlowJo Software (BD Life Sciences, Franklin Lakes, NJ, USA).

T cell responses to TBEV were assessed using multi-color flow cytometry, and the monoclonal antibodies (mAbs) used were; anti-CD107a FITC, anti-CD4 Pacific Blue, anti-CD8 PerCP, anti-HLA-DR PerCP, anti-Ki67 FITC, anti-Ki67 Alexa Fluor 700, anti-Bcl2 PE, anti-CCR7 PE-Cy7, anti-MIP-1β PE, anti-CD14 BD horizon V500, anti-CD19 BD horizon V500, anti-perforin FITC and anti-granzyme B APC, anti-granzyme B PE-CF594 all from BD Biosciences (San Jose, CA). Anti-CD45RA APC-Cy7, anti TNF pacific blue, anti-IFN-γ Brilliant Violet 570, anti-CD27 Brilliant Violet 785, anti-CD27 Brilliant Violet 421, anti-Helios Pacific Blue, anti-T-bet Alexa Fluor 488, anti-T-bet PE-Cy7, anti-CCR7 Brilliant Violet 785, anti-CD279 Brilliant Violet 711, anti-CD27 biotin and anti-CD127 Brilliant Violet 570 were all from Biolegend (San Diego, CA). Anti-CD38 Alexa Fluor 700, anti-CD38 eFluor 650, anti-CD127 Alexa Fluor 780, anti-PD-1 (CD279) PE, anti-Eomes eFluor 660 and IgM eFluor 650 were all from eBioscience (San Diego, CA). Anti-CD4 Qdot 605, anti-CD8 Qdot705, anti-CD8 Qdot 605, Streptavidin-Qdot 585, anti-CD57 pure and Aqua Live/Dead were all from Invitrogen (Carlsbad, CA). Anti-CD3 ECD, anti-CD3 PE-Cy5, HLA-A2 CMV pp65 tetramer in PE and anti-CD56 ECD were from Beckman Coulter (Brea, CA). HLA-A2 ILLDNITTL tetramer in PE was kindly provided by the NIH Tetramer core facility.