Fitc labeled anti cd56

After 7 days of expansion, T cells were harvested and immunophenotyping was performed. T cells were incubated with the following monoclonal antibodies: fluorescein isothiocyanate (FITC)-, phycoerythrin (PE)-, PE-cyanine 7 (PE-Cy7)-, and V450-labeled anti-CD3; allophycocyanin (APC)-, and Alexa Fluor 700-labeled anti-CD4; Peridinin-chloropyll-protein complex (PerCP)-labeled anti-7-Aminoactinomycin D (7-AAD); FITC-labeled anti-CD28; FITC-labeled anti-CD69; FITC-labeled anti-CD94; FITC-labeled anti-CD56; FITC-labeled anti-TCRαβ; FITC-labeled anti-CD95; APC-labeled anti-CD45RO; PE-Cy7-labeled anti-CCR7; FITC-labeled anti-CD25; APC-labeled Annexin V (all from BD Bioscience, Franklin Lakes, NJ, USA); FITC-labeled anti-TCRγδ (Becman Coulter, Fullerton, CA, USA); APC-Alexa Fluor 700-labeled anti-CD127 (Beckman Coulter, Fullerton, CA, USA); Pacific Orange-labeled anti-CD8 (Invitrogen); PE-labeled anti-CD39 (BD Bioscience, Franklin lakes, NJ, USA) for 15 min and washed before flow cytometry (FACS Aria flow cytometer, BD Biosciences) and analysis (FlowJo software, Tree Star, Inc., Ashland, OR, USA). T cell death was assessed by flow cytometry staining for 7-AAD and Annexin V after 7 days culture with beads and cytokines followed by a brief (6 h) period of stimulation with peptide mix spanning the cytomegalovirus (CMV) protein pp65.

Dextran (50,000), Dextran sulfate (7000–20,000), chondroitin sulfate, heparin, N-acetyl heparin, and trypsin-EDTA (0.25%) were purchased from Sigma-Aldrich (St. Louis, MO, USA), and the powder reagents were dissolved in sterile water. FITC- or PE-labeled anti-CD138, anti-CD4, anti-CD19, and monensin were purchased from Biolegend (San Diego, CA, USA). FITC-labeled anti-CD56 and PE-Cy5-mouse anti-human CD107a were purchased from BD Pharmingen (San Diego, CA, USA). FITC-labeled anti-human or anti-mouse IgG Fc was purchased from Sigma (St. Louis, MO, USA). PE-conjugated streptavidin was purchased from BD Biosciences (San Jose, CA, USA). Cell trace carboxyfluorescein succinimidyl ester (CFSE) was purchased from Invitrogen (Carlsbad, CA, USA). Protein A/G agarose and heparin sepharose were purchased from Santa Cruz Biotechnology (Dallas, TX, USA) and Abcam (Cambridge, UK), respectively.