Anti cd3 ucht1

Monoclonal antibodies, conjugated with different fluoro-chromes, were used to stain cell surface markers including: anti-CD3 (UCHT1; R&D, Minneapolis, MN, USA), anti-CD20 (clone 2H7; BD Biosciences), anti- KI67(clone: 20Raj1, eBioscience, CA 92121, USA), anti-caspase3 (Cat:51-68655X; BD Biosciences) and TdT (cat:51-35404X.4; BD Biosciences). Matched isotype control with appropriate fluro-chrome was used. Propidium Iodide (1.0mg PI/ml; Invitrogen) was used to exclude dead cells from analysis. Cells were incubated with related antibodies for 20 minutes. Afterwards samples being analyzed on FACSCalibur (BD Bioscience), between 30000 and 80000 events were collected and data analysis was performed using FlowJo software.

Anti-CD3 (UCHT1) and PE conjugated anti-SLAMF6 (292811) were purchased from R&D Systems. Goat-anti-mouse IgG (Poly4053), Alexa Fluor 647 conjugated anti-ERK (4B11B69), phospho Thr202/Tyr204, Alexa Fluor 647 conjugated IgG2b iso-type control (MOPC-173), and anti-SLAMF6 (NT-7) were purchased from BioLegend. SLAMF6 (HA12OC3104) was purchased from Sino Biologics Inc. PE conjugated IgG2a iso-type control (eBR2a) was purchased from eBioscience. Western blot antibodies for pERK (E10) Thr202/Tyr204, pZAP70 (Y352) Tyr309, pAKT (L32A4) Thr308 and pSRC (Y527) were purchased from Cell Signaling. Anti-beta actin (AC-15) was purchased from Invitrogen. Western blot secondary antibodies IRDye 680RD Goat anti-mouse IgG, IRDye 800CW Goat anti-mouse IgG and IRDye 680RD Goat anti-rabbit IgG were purchased from Licor. Anti-SLAMF6 (NT-7) Fab fragments were generated using the Pierce Fab Preparation Kit obtained from Thermo Fisher. Cells were stimulated with the following soluble antibodies: anti-CD3/anti-mouse IgG (3.25 μg/mL), anti-SLAMF6 (3.25 μg/mL), anti-mouse IgG (1.63 μg/mL), anti-SLAMF6 Fab (3.25 μg/mL) anti-mouse IgG (1.63 μg/mL). Cells were stimulated with the following immobilized antibodies: anti-CD3/anti-mouse IgG (1.5 μg/mL), anti-SLAMF6 (5 μg/mL).

Primary T cell were isolated from unidentified donors through New York Blood Center. Consenting is not applicable. Isolation was done using RosetteSep (StemCell) followed by Ficoll-Paque. The cells were maintained in enriched media (Hepes 25 mM, sodium pyruvate 100 mM, 1% nonessential amino acids, and 1% l-glutamine) at 5% CO₂ and at 37 °C. Primary murine splenocytes were isolated by mechanical disruption of spleens from 10 to 12 week-old mice to generate a single cell suspension. Primary murine CD8⁺ T cells were isolated from spleens of 10–12 week-old mice by CD8⁺ negative selection (StemCell). Jurkat T cells were obtained from the ATCC and maintained in RPMI medium supplemented with 10% FBS and 1% Pen/Strep (10,000 U/ml stock). Constructs were introduced into the cells by nucleofection (Lonza), efficiency of 50–70%. Cells were stimulated at a 1:3 ratio with magnetic beads conjugated with anti-CD3 (UCHT1; R&D) and IgG1 (R&D) or with anti-CD3 and PDL2-IgG1 (R&D) or with anti-CD3 and PDL1-IgG1 (R&D)^{33 (link)}. magnetic beads (Invitrogen) were coated with anti-CD3 (25%) and PDL2-Ig or PDL1-Ig fusion protein (50%); control IgG comprised the remaining total protein.