Quantifying CAR1 and CD16 Expression in Engineered NK Cells

RT-PCR and ELISA were carried out to verify expression of CAR1 and CD16 on lentivirus-transduced or plasmid-transfected cells. Total RNAs were extracted from NK92MI CAR1 dimer, monomer and untransduced cells using TriZol Reagent (Invitrogen). RT-PCR for CAR1, CD16 and beta actin was performed using One-Step RT-PCR kit (New England Biolabs) with corresponding primers (Supplementary Table S1) and was analyzed by agarose gel electrophoresis. RIPA lysis buffer (Santa Cruz) and M-PER Extraction buffer (Thermo Fisher) were used to extract whole cell lysates from NK92MI untransduced and lenti-transduced CAR1 dimer, monomer and GFP only cells. Protease inhibitors (Santa Cruz and Sigma) were added to RIPA (Santa Cruz) and M-PER (Thermo Scientific) lysis buffers, respectively, prior to use. Protein concentrations in the cell lysates were determined by Protein Assay Reagent (Bio-Rad) using Bovine Serum Albumin standard protein (Pierce). CAR1 concentration was assayed by Paired Human Factor VII Antibody Sandwich ELISA (Cedarlane Laboratories) using recombinant L-ICON1 protein as standards following the published procedure with a minor modification (1:200 dilution for capture and detection Abs)^{14 (link)}. CAR1 expression was normalized and presented as ng per μg cell lysate.