Live dead fixable far red dead cell staining kit

CD4+ T cells were obtained from peripheral blood from healthy volunteers. Blood samples were diluted 1:2 with PBS before adding 50 μL/mL of RosettSep Human CD4+ T cell enrichment cocktail (STEM CELL) for 20 min at room temperature. After incubation, samples were diluted again 1:4 with PBS and CD4+ T cells were obtained directly from the mononuclear fraction obtained after Ficoll–Hypaque density gradient centrifugation. CD4+ T cells were washed twice, counted, and resuspended in PBS. CD4+ T cells were then stained with 1 μM Cell Trace Violet Cell Proliferation Kit (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) for 20 min at 37 °C, protected from light. Unbound dye was quenched by adding cold FCS for 5 min at 4 °C. CD4+ T cells were washed two times and resuspended in X–VIVO (Gibco) with IL–2 (50 U/mL, ProLeukin). Cells (2 × 10⁵) were activated for 48 h with CD3/CD28 Dynabeads (Invitrogen) prior to adding the compounds. After 48 h, compounds were added (EC₂₅) and incubated for four days. To analyze proliferation and viability, cells were stained with Live/Dead fixable far red dead cell staining kit (Invitrogen) for 30 min at 4 °C and counting beads were added before acquisition. Cells were acquired on an LSRFortessa X20 (BD) within 24 h. Data were analyzed using FlowJo (Tree Star, Inc., Ashland, OR, USA).