2.5 Quantification of target expression levels on cells
PD-L1-transduced CHO K1 cells, H2444 cells, and MDA-MB-231 cells were
all quantified for PD-L1 expression using Quantum Simply Cellular
anti-mouse IgG beads (Bangs Laboratories), according to the
manufacturer’s protocol. Standard beads were incubated with an
APC-conjugated mouse anti-human PD-L1 antibody (BioLegend, clone MIH2)
(1:50 dilution) in PBSA for 30 min at room temperature. The beads were
then pelleted, washed, resuspended in PBSA, and analyzed via flow
cytometry to generate a standard curve. Cells were grown to 60-90%
confluency and detached with trypsin-EDTA (Thermo Fisher Scientific) and
quenched via addition of culture medium. 2×105 cells
were then aliquoted into microcentrifuge tubes, washed, and resuspended
in PBSA containing APC-conjugated mouse anti-human PD-L1 antibody
(BioLegend, clone MIH2) (1:50 dilution) and incubated for 30 min at 4°C
with rotation. Finally, the cells were washed, resuspended in PBSA, and
analyzed via flow cytometry. Mean fluorescence intensity (MFI) values
were compared to the generated calibration curve to determine PD-L1
expression levels.