Celltrace violet ctv dye

PBMC were resuspended at a concentration of 10 x 10⁶/ml in PBS and incubated with Cell Trace Violet (CTV) dye (Thermofisher) at 5 µM for 20 min at RT, according to the manufacturer’s directions. Cells were washed once with 5x volume of IMDM/10% human serum and resuspended for cultures of 300,000 PBMC in 200 µl/well of a 96-well plate and incubated for 7 days in a 5% CO₂ incubator. Different wells contained different antigens as indicated. All experiments also included: (i) culture medium only negative control well; (ii) anti-CD3/anti-CD28/anti-CD2 T cell activator (1/200 dilution) polyclonal positive control well; and (iii) influenza virus (1/200 dilution) antigen positive control well. After 7 days, cells from the respective cultures were stained with CD3-PerCP-Cy5.5, CD4-FITC, CD8-APC-H7 and CD25-APC (BD Biosciences), and analysed on a 5-laser Fortessa X20 (32 (link)) and antigen-specific CD4 T cells gated as CD3+CD4+CD25^highCTV^dim as previously described (34 (link)). Cultures were classified as positive for antigen-specific CD4 T cells if the CD25^highCTV^dim % of CD4+ CD3+ T cells was ≥ 1%.

Human T cells were isolated from PBMC using EasySep™ Human CD4⁺ or CD8⁺ T Cell Isolation Kit (STEMCELL Technologies) according to the manufacturer’s protocols. For cell proliferation assay, T cells were labeled with Cell Trace Violet (CTV) dye (Thermo Fisher Scientific) for 20 min at 37 °C at a final concentration of 2.5 μM in PBS. Next, the labeled T cells were plated in round-bottomed 96-well plates (2 × 10⁴ cells per well) in ʟ-arginine-free RPMI medium (SILAC RPMI medium, Thermo Fisher Scientific) supplemented with 10% (v/v) dialyzed FBS (Thermo Fisher Scientific), 2 mM glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, 1% (v/v) MEM non-essential amino acids solution (Thermo Fisher Scientific), 50 μM 2-mercaptoethanol (Thermo Fisher Scientific), and 150 µM ʟ-arginine and 40 mg/l ʟ-lysine (Sigma-Aldrich). Proliferation was triggered by the stimulation with Dynabeads Human T-Activator CD3/CD28 (ratio 1:2, Thermo Fisher Scientific). T-cells were co-cultured with BM stromal cells in T cells to BM cells ratios 1:0.3, 1:1, 1:3, 1:10^{9 (link),22 (link)}.

Thawed autologous PHA-blasts were cultured in RPMI1640 medium containing 10% FBS and 100 U/mL IL-2 in 24-well plate for 1 to 3 days prior to use in a cytotoxic assay to evaluate autoimmune effect by activated autologous T cells. Collected PHA-blasts were stained as target cells with 2.5 μM of CellTrace Violet (CTV) dye (ThermoFisher Scientific, C34557) for flow cytometry analysis. T cells with peptide-loaded DCs or non-peptide loaded DCs were cultured for at least 2 h prior to the assay. Those T cells were co-cultured with stained PHA-blasts in the effector to target cells (E:T) ratio of 20:1 in 6-well plate at 37 °C for 4 h^{21 (link)}. PHA-blasts without T cells were also incubated in the same manner to detect basal cell death for the calculation of specific cell lysis by T cells. After the incubation, cells were stained with eBioscience Fixable Viability Dye (FVD) eFluor 780 (Thermo Fisher Scientific, 65–0865) and fixed with 4% formaldehyde, followed by filtering through 70 μm and 40 μm cell strainers. Single cell suspension was transferred to FACS tube and stored at 4 °C protected from light until use in flow cytometry analysis. Percentages of dead PHA-blast cells (CTV-positive/FVD-positive) were measured by BD LSRFortessa flow cytometry. The percentage of specific lysis was calculated by the same equation used for cytotoxicity assay^{58 (link)}.

To monitor cell division, purified B cells were labeled with 2.5 μM CellTrace Violet (CTV) dye (Thermo Fisher Scientific, Waltham, MA, USA) in PBS at 37 °C for 20 min according to the manufacturer’s protocol prior to stimulation. Cell divisions were analyzed with the proliferation platform of FlowJo v10.7.2 software (Tree Star, Ashland, OR, USA).