Anti cd20 apc

Wildtype JeKo-1 cells were seeded at 1 × 10⁴ cells in 100 μl per well of complete media in 96 well round bottom plates. CAR-19 T cells in 100 μl complete media were added to give E:T cell ratios of 1:1, 0.3:1, or 0.1:1 and the co-culture was incubated for up to 13 days. Samples were evaluated at days 1, 6, 11, and 13 for the accumulation of a CD19-negative/CD20-positive population, using flow cytometry (FMC63-PE, and anti-CD20-APC, BD). Once a CD19-negative population emerged, the lymphoma cells were counted and replated at 1 × 10⁴ cells in 50 μl complete media. JeKo-1 cells were left untreated in media, or were incubated with 1 × 10⁵ CAR-19 T cells added in 50 μl complete media (E:T ratio of 10:1), with or without CTE19.20-His protein in 50 μl complete media. In a set of control wells the CTE19.20-His protein was added to the JeKo-1 cells alone, without CAR-19 T cells. In each case, the 150 μl co-culture was incubated for 48 hours. Then, the JeKo-1 cells were stained and analyzed by flow cytometry using HIB-CD19-PE (BioLegend) and anti-CD20-APC for cells not incubated with protein or with anti-CD20-APC and anti-ROR1-PE (BioLegend) for samples treated with CTE-19.20 protein. 7AAD was used to gate out dead cells and doublets were excluded using FSC height versus area and gating accordingly.