Ki67 bv421 clone b56

EBV-associated cancer cells were plated at a density of 1×10⁵ cells/well. After 24 hours, T cells were added at an effector-to-target ratio of 50:1 and the culture was incubated for 24 hours at 37°C and 6.5% CO₂. To assess the impact of T cells on cancer cells, the cultured cells were then incubated at 4°C with the following antibodies: human anti-CD45-V500 (clone HI30, BD Biosciences), anti-CD3-AF700 (clone HIT3a, BioLegend), anti-CD56-BV421, anti-CD8-PerCP-Cy5.5, anti-CD19-APC-Cy7, anti-perforin-PE (clone dG9, eBioscience), anti-granzyme K-FITC (clone G3H69, BD Biosciences), anti-granzyme B-BV711 (clone GB11, BD Biosciences), Ki67-BV421 (clone B56, BD Biosciences), anti-active caspase-3-BV605 (clone C92-605, BD Biosciences) and LIVE/DEAD Fixable Near-IR Dead Cell Stain (Thermo Fisher Scientific, MA). Flow cytometry was performed using a BD LSRFortessa with FACSDiva software and postacquisition analysis was performed using FlowJo software.

Intranuclear expressions of Bcl-6, T-bet, and Ki67 were evaluated by flow cytometry. First, 5x10⁶ splenocytes were stained for surface markers and viability, as previously described. Then, cells were fixed and permeabilized with the FOXP3 labeling kit (eBioscience), according to the manufacturer’s instructions. Anti-Bcl6-PE (clone K112-91, BD Biosciences), and/or anti-T-bet-BV421 (clone O4-46, BD Biosciences), and/or KI67-BV421 (clone B56, BD Biosciences) were added and incubated for 1 hour on ice and in the dark. Cells were washed twice in FACS buffer and one million total events were acquired.