Working solution of CellTrace Far Red is a 1:1000 dilution of CellTrace Far Red DMSO stock solution (1 mM) in pre-warmed PBS. Working solution was prepared immediately prior to use.
Isolated T cells were incubated with CellTrace Far Red working solution (10^6 cells / mL staining solution) for 20 min at 37°C in the dark. To remove free dye, R10 medium (5 × staining solution volume) was then added to the cells for an additional 5 min. The cell solution was centrifuged (524 × g, 5 min, 4 °C) and the supernatant was removed. The remaining pellet was resuspended in pre-warmed R10 medium.
The stained T cells were then cultured for 4 days as stated insection 2.3 . As described in section 2.4 , the cells were stained for viability and for surface markers, and analyzed via flow cytometry, with the following two modifications: The only antibodies used for surface staining were CD3 FITC (clone HIT3a) and CD4 PerCP-Cy5.5 (clone RPA-T4) (both from Biolegend). A 1:1 mixture of unstained T cells and CellTrace Far Red stained T cells was used to establish compensation settings.
Isolated T cells were incubated with CellTrace Far Red working solution (10^6 cells / mL staining solution) for 20 min at 37°C in the dark. To remove free dye, R10 medium (5 × staining solution volume) was then added to the cells for an additional 5 min. The cell solution was centrifuged (524 × g, 5 min, 4 °C) and the supernatant was removed. The remaining pellet was resuspended in pre-warmed R10 medium.
The stained T cells were then cultured for 4 days as stated in