Anti cd19 pb clone hib19

At harvest or upon thawing, 1 × 10⁶ cells were stained for cell surface markers to analyze T-cell differentiation status. The following pretitrated antibodies were used: anti-CCR7–FITC (clone 150503; BD Pharmingen); anti-CD45RO–PE (clone UCHL1), anti-CD8–H7APC (clone SK1; BD Biosciences); anti-CD95–PerCP-Cy5.5 (Clone DX2), anti-CD4–BV510 (clone OKT4), anti-CD3–BV605 (clone OKT3), anti-CD14–Pacific Blue (PB; clone HCD14), anti-CD19–PB (clone HIB19; BioLegend); anti-CD27–PE-Cy7 (clone 1A4CD27; Beckman Coulter); and carboxyfluorescein diacetate succinimidyl ester (CFSE) and ViViD (Invitrogen). The anti-CAR19 idiotype for surface expression of CAR19 was provided by Novartis (Basel, Switzerland).
Cells were washed with PBS and stained for viability using LIVE/DEAD Fixable Violet (Molecular Probes) for 15 minutes, washed once, and resuspended in fluorescence-activated cell sorting (FACS) buffer consisting of PBS, 1% BSA, and 5 mmol/L ethylenediaminetetraacetic acid (EDTA). Cells were then incubated with the above indicated antibodies for 1 hour at 4°C. Sample were then washed 3 times with FACS buffer and fixed in 1% paraformaldehyde. Positively stained cells were differentiated from background using fluorescence-minus-one controls. Flow cytometery was performed on BD LSR Fortessa. Analysis was performed using Flowjo software (Tree Star Inc. version 10.1).