Violet proliferation dye

Prior to stimulation for B10 cells, B cells were negatively isolated from a starting population of 10⁷ PBMCs, using the B cell enrichment kit (Stemcell). Isolated B cells were plated in 96-well flat bottom plates in RPMI + 10% FBS. Cells were stimulated with CpG and rCD40L for 48 h at 37°C in 5% CO₂ incubator. At 43 h, cells were restimulated with PMA and ionomycin. In parallel with the end of the 48-h stimulation, PBMCs from the same patients were enriched for CD4 T cells (Stemcell) and stained with the Violet Proliferation dye (1 µM, BD Bioscience) in PBS and incubated in a 37°C water bath for 15 min. After 48 h, the isolated CD4 cells were combined with B cells at 1:1 and 1:2 ratio of T:B cells along with a CD4 T cell only condition and stimulated with αCD3/αCD28 for 5 days. For experiments using the transwell plates, stimulated B cells were plated in top chamber of 24-well transwell plates while CD4 T cells and αCD3/αCD28 were plated in bottom chamber.
To visualize proliferation of the CD4 T cells, cells were stained with Zombie Violet, CD14 Brilliant Violet 510 (M5E2, Biolegend), CXCR5 AlexaFluor 647 (RF8B2, BDB), CD8 AlexaFluor 700 (SK1, Biolegend), CD3 APC-Cy7 (SK7, Biolegend), CD19 PE (HIB19, Biolegend), and CD4 PE-Cy7 (SK3, Biolegend) conjugate for 25 min at 4°C. Following cell surface staining, cells were fixed with 1% PFA and acquired on a LSRII flow cytometer (BDB).

CD8 T cells were isolated from an OT1+ mouse using the mojosort mouse CD8 T cell isolation kit (Biolegend) and labeled with violet proliferation dye (BD Biosciences cat# 562158). For DC-T cell co-culture, BMDCs were treated with psOVA (5 μg), or ova+psDNA (5 μg) for 1, 3, or 7 days. BMDCs were washed and then co-cultured with labeled OT1s for 3 days at a 1:10 ratio of BMDC:OT1. Cells were then stained and run on a flow cytometer. OT1 division (percent dividing cells) was calculated as previously described (Roederer, 2011 (link)) using the equation fraction diluted $={\sum }_1^i\frac{N_i}{2^i}\mathrm{}/{\sum }_0^i\frac{N_i}{2^i}$ , where i is the generation number (0 is the undivided population), and N_i is the number of events in generation i.