Cd3 ax700

Cryopreserved PBMC/cord BMC (CBMC) were thawed, counted, and processed immediately for phenotypic assessment using two staining panels. Panel A consisted of surface staining with Zombie yellow (viability), CD25 FITC (BioLegend), Lag3 PE (Invitrogen), CTLA4 PE-CF594 (BD Biosciences), CD4 PerCP-Cy5.5 (BD Biosciences), CD3 Ax700 (BD Biosciences) followed by fixation and permeabilization using the eBioscience Foxp3 / Transcription Factor Staining Buffer Set (eBioscience). Intracellular staining was then performed with FoxP3 Ax647 (BD Biosciences), Granzyme B APC-fire750 (BioLegend), IL-10 BV421 (BioLegend) and TGFβ PE-Cy7 (BioLegend). Panel B consisted of surface staining with Zombie yellow (viability), CD4 FITC (BioLegend), CD3 PE-CF594 (BD Biosciences), GITR PerCP-Cy5.5 (BioLegend), TNFR2 PE-Cy7 (BioLegend), CD39 Ax700 (R&D Systems), PD1 APC-Cy7 (BioLegend) and TIGIT BV421 (BioLegend). Intracellular staining consisted of FoxP3 Ax647 (BD Biosciences) and IL-35 PE (BioLegend). Analysis was performed using the Galios instrument (Beckman Coulter).

PBMC were isolated and cryopreserved following standard procedures [33 (link)], then shipped to the FHCRC laboratory in Seattle for later analysis. To evaluate T-cell activation, cryopreserved PBMC were thawed in R10 (RPMI 1640 supplemented with 2mML-glutamine, 25mM HEPES (Invitrogen) 10% FBS (Benchmark), 50μg/ml streptomycin, 50U/ml penicillin (Invitrogen), and 50U/ml benzonase (Novagen), and then washed and stained immediately. Sample viability was determined by LIVE/DEAD Aqua Cell Stain (Invitrogen). Surface staining used integrin β7 Phycoerythrin Cyanine (PECy) 5, Cluster of differentiation (CD) 49d (α4) Allophycocyanin (APC), C-C chemokine receptor type 5 (CCR5) PECy7, Cutaneous Leukocyte Antigen (CLA) biotin, CD14 V450 (all Becton Dickinson [BD] Biosciences), and CD103 (αE) Peridinin Chlorophyll Protein Cyanine (PerCPCy) 5.5 (Acris Antibodies). Intracellular staining used CD3 Ax700, Ki-67 Fluorescein Isothiocyanate FITC, B cell lymphoma 2 (Bcl-2) PE, Streptavidin APC-Cy7 (all BD), and CD4 Energy Coupling Dye ECD (Beckman Coulter). Flow cytometric analysis was performed using an LSR II flow cytometer (BD); post-acquisition analysis used FlowJo software (Tree Star, Inc). Personnel involved in thawing, staining and acquiring flow cytometric data were blinded from samples’ pre/post procedure status.