Live dead fixable near ir viability dye

CAR-meGFP-transduced Jurkat cell clones with stably low CAR expression (“low clone” and “mid clone”) were spiked into mCherry-transduced non-CAR Jurkat cell at ~0.2% prevalence. Subsequently, cells were stained with 3 nM of allophycocyanin (APC)-labeled CD19-tetramers or CD19-dodecamers for 1 hour at 4°C in FACS buffer (PBS, 2% BSA, 0.05% sodium azide). Stained cells were washed three times and then incubated with anti-APC microbeads (Miltenyi Biotec, 130-090-855) at 1:5 dilution for 15 minutes at 4°C. Cells were then washed once with FACS buffer and applied onto magnetic columns (Miltenyi Biotec, 130-042-201). Columns were washed with three column volumes of FACS buffer. Then, the column was removed from the magnet, and cells were eluted under pressure from a plunger. Subsequently, cells were centrifuged and incubated briefly with LIVE/DEAD Fixable Near-IR viability dye (Invitrogen, L34975) diluted 1:1000 in PBS for 5 minutes at room temperature. Finally, cells were washed once in FACS buffer at 4°C before analysis by flow cytometry.

Cryopreserved patient biospecimens were thawed in warm RPMI+10% FBS. Cells were first washed with cold FACS buffer (PBS, 2% BSA, 0.05% sodium azide). Next, Fc receptors were blocked by incubation with Human TruStain FcX (BioLegend, 422301) at 1:50 dilution for 5 minutes at 4°C. Then, cells were incubated for 30 minutes at 4°C in the dark with a staining solution containing BV421-anti-CD3ε (clone SK7, BioLegend, 344833), PE-anti-CD4 (clone SK3, BioLegend, 344605), AF488-anti-CD8α (clone Hit8a, BioLegend, 300916), and either antigen-multimers or biotinylated anti-FMC63 (clone Y45, Acro Biosystems, FM3-BY45). For staining with biotinylated anti-FMC63, cells were subsequently incubated with 1 ng/μL AF647-streptavidin (BioLegend, 405237) in FACS buffer for 20 minutes. Monoclonal antibodies were generally used according to manufacturer recommendations.
After staining, cells were incubated briefly with LIVE/DEAD Fixable Near-IR viability dye (Invitrogen, L34975) diluted 1:1000 in PBS for 5 minutes at room temperature. Then, cells were three times in FACS buffer at 4°C before analysis by flow cytometry.

CAR-transduced Jurkat cells were incubated in T-cell media (RPMI supplemented with 10% FBS, 1% Pen/Strep, 2 mM L-glutamine, 50 mM 2-mercaptoethanol) with antigen-multimers or anti-idiotype antibodies. After 24 hours at 37°C or 4°C, cells and supernatants were collected for the analyses of activation markers and cytokine production respectively.
To measure T-cell activation markers, the cells were washed with FACS buffer (PBS, 2% BSA, 0.05% sodium azide) and incubated for 30 minutes at 4°C in the dark with a staining solution containing APC-anti-CD25 (clone M-A251, BioLegend, 356110) and BV510-anti-CD69 (clone FN90, BioLegend, 310936). Subsequently, cells were incubated with LIVE/DEAD Fixable Near-IR viability dye (Invitrogen, L34975) diluted 1:1000 in PBS for 5 minutes at room temperature. Finally, cells were washed three times in cold FACS buffer before analysis by flow cytometry.
The supernatant was diluted 1:2 in PBS and analyzed for IL-2 secretion by an enzyme-linked immunosorbent assay (IL-2 Quantikine ELISA Kit, R&D Systems, D2050). For each ELISA, an 8-point standard curve was generated (R²> 99%).