Cd3 buv737

BMMC were stained with the following anti-human antibodies: IgD-Brilliant Violet 480 (Cat. #566138; BD Biosciences), CD3-BUV737 (Cat. #612750; BD Biosciences), CD14-BUV737 (Cat. #612763; BD Biosciences), CD19-Spark NIR 685 (Cat. #302270; BioLegend), CD38-Brilliant Violet 785 (Cat. #303530; BioLegend), CD138-APC-R700 (Cat. #566050; BD Biosciences), CD27-Brilliant Violet 711 (Cat. #356430; BioLegend), CD134 (OX40)-Brilliant Violet 510 (Cat. #350025; BioLegend), CD246 (ALK)-Alexa Fluor 488 (Cat. #NBP3–08771AF488; Novus), CD357 (GITR)-Brilliant Violet 605 (Cat. #747664; BD Biosciences), and CD45-PE-Cy5 (Cat. #304009; BioLegend). Samples were run on a Cytek’s Aurora Spectral Flow Cytometer analyzed with the FlowJo v10.8.1 software (FlowJo, LLC).

Lymph node cell suspensions from influenza infected ferret were stained with the following panel: live/dead Blue (Thermo Fisher), CD79a PerCP-Cy5.5 (HM47; BioLegend)^{24 (link)}, CD8 AF700 (OKT8; Thermo)^{24 (link)}, CD4 FITC (from CSIRO)^{25 (link)}, BCL6 AF647 (K112-91; BD), BCL6 AF647 (IG191E/A8; BioLegend), CXCR5 BV421 (L138D7; BioLegend), CXCR5 BB515 (RF8B2; BD), CXCR5 PE (2G8, BD); CXCR5 Biotin (in-house), Streptavidin BV421 (BD), PD-1 BV786 (29F.1A12; BioLegend), PD-1 BV421 (EH12.2H7; BioLegend), PD-1 PE (in-house). For BCL6 staining, cells were fixed, permeabilized, and stained using the BD Transcription Factor Buffer kit (BD) according to the manufacturer’s instructions. Macaque LN suspensions were stained with Live/dead Aqua (Thermo Fisher), CD4 BV605 (L200; BD), CXCR5 PECy7 (MU5UBEE, Thermo Fisher), and CD3 BUV737 (SP34-2, BD). All samples were acquired on a BD LSR Fortessa using BD FACS Diva and data was analyzed in FlowJo v10.

Cryopreserved health donor PBMC were thawed briefly in a 37 °C water bath. CD8⁺ T cells were enriched using magnetic beads (Miltenyi Biotec). Cells were washed by centrifugation and then treated with PBS (Gibco, #14190-250) containing benzonase (Millipore, #70664) and 50 nM Dasatinib (Axon Medchem, #1392) for 45 min at 37 °C. Cells were transferred to a 96-well assay block (Corning, #3960), centrifuged, and supernatant was aspirated. The appropriate custom Immudex dCODE-PE dextramer pool (Copenhagen, Denmark) was added at 1 µl/100 µl reaction for 30 min in dark at room temperature. Next, the fluorochrome-labeled surface markers were added, and the cells were incubated for additional 30 min at 4 °C. After washes, the cells were immediately sorted. Flow cytometry antibody staining, and washes were performed in staining buffer (BD, #554657). Surface markers for FACS included the following markers and fluorophores: Live/Dead—DAPI (Sigma, #10236276001), CD3 BUV737 (BD Biosciences, #612750), CD4 BV510 (BD Biosciences, #563919), CD8 BUV805 (BD Biosciences, #612889), CCR7 AF647 (BioLegend #353218), and CD45RO BV605 (BioLegend #304238).

Thymuses from WT or Yap-cKO mice (8–10 weeks old) were mechanically disrupted by being pushed through a 70 μm mesh (Falcon) with an insulin syringe plunger and washed with PBS. Cells were treated with ACK red blood cell lysis buffer, and thymocyte single-cell suspensions were stained for analysis by flow cytometry as described earlier. Cells were stained with dead cell dye and antibodies for the following surface markers: CD3 BUV737 (BD), CD4 BUV395 (BD), CD8 PerCP-Cy5.5 (Biolegend), TCRβ BV510 (Biolegend), CCR7 PECy7 (Biolegend), H-2Kb PE (Biolegend), CD69 BV421 (Biolegend), CD45R/B220 APCFire750 (Biolegend), CD25 APCFire750 (Biolegend), GL3 APCFire750 (Biolegend), and NK1.1 APCFire750 (Biolegend). Thymocytes were also stained intracellularly with anti-Nur77 APC (BD) using the eBioscience Foxp3/Transcription factor staining buffer set, as described earlier.

Cells were cultured and stimulated in 96-well plates at 1 × 10⁵ cells per well. Plates were coated with anti-CD3 antibody (Biolegend) at concentrations of 0, 0.125, 0.25, 0.5, and 1 μg/ml at 4°C overnight and were washed twice with PBS before incubation. Cells were stimulated in the anti-CD3–coated plates with soluble anti-CD28 at 2 μg/ml (Biolegend). On days 1 and 3, cells were stained with dead cell dye as well as antibodies recognizing the lineage and activation markers CD3 BUV737 (BD), CD4 BUV395 (BD), CD8 PerCP-Cy5.5 (Biolegend), CD69 PE (Biolegend), CD44 BV650 (Biolegend), and CD25 APC (Biolegend). For proliferation assays, CD4⁺ or CD8⁺ T cells were isolated and stained using the CellTrace Violet or CFSE Cell Proliferation Kit (Life Technologies). Briefly, purified cells were washed with PBS and incubated with CellTrace dye for 20 minutes at 37°C protected from light. After 20 minutes, complete RMPI medium was added to the cell suspension, and the cells were incubated 5 minutes further before being washed and resuspended in complete RPMI medium. Cells were cultured in 96-well plates at 1 × 10⁵ cells per well and were stimulated using anti-CD3/CD28 dynabeads (Gibco) at a 1:1 ratio with T cells. On days 1 and 3, cells were stained with dead cell dye, and proliferation was measured at the same time.