Anti cd3 antibody clone ucht1

This was as described in (19 (link)). In brief, cultured CTLs were added to the P815-NucLight Red-expressing target cells in the presence or absence of 1 μg/ml anti-CD3 antibody (clone UCHT1, catalogue 555330, BD Biosciences—Pharmingen), at a CTL-to-target ratio of 10:1. The killing was measured by the reduction of red fluorescence intensity, indicating target cell death. The assay was measured every 30 min for 4 h using the IncuCyte S3 Live-Cell Analysis System (Essen Bioscience).

CTLs and P815 target cells were resuspended in RPMI (without phenol red, Gibco), 2% FBS, and 0.5 μg/mL anti-CD3 antibody (clone UCHT1, BD Biosciences); plated at different effector/target ratios as shown; and incubated at 37°C for 4 hours. The CytoTox 96 kit (Promega, Madison, Wis) was used to assess target cell lysis per the manufacturer's instructions. Activated purified NK cells were tested for cytolytic activity against the 221 B-EBV target cell line in a standard ⁵¹Cr-release assay.¹¹ (link)

Flat bottom 96-well plates (3595, Corning) were coated with 50 μL/well Poly-l-ornithine (PLO) (P4957, MilliporeSigma) for 1 hour at room temperature (RT) and subsequently dried for 1 hour at RT. P815-NucLight Red (2500 cells) were seeded per well in phenol red–free human T cell media without IL-2 and left to attach to the plate overnight at 37°C. Additionally, hCTLs (day 11 after stimulation) were cultured in human T cell media without IL-2 overnight at 37°C. The following day, hCTLs were added to the P815-NucLight Red target cells in the presence or absence of 0.5 μg/mL anti-CD3 antibody (clone UCHT1, catalog 555330, BD Biosciences — Pharmingen) at an effector-to-target ratio of 8:1. Loss of red fluorescence (i.e., targeted cell death) was monitored every 30 minutes for 8 hours using the IncuCyte S3 live-cell analysis system (Essen Bioscience) and plotted as percentage of lysis.