Ique3 high throughput flow cytometer

BV2-LILRB2 or HMC3 cells were detached from culture plates by non-enzymatic dissociation buffer (Thermo Fisher Scientific). A total of 1 million LILRB2-BV2 or HMC3 cells were blocked by 0.1 mg/mL human Fc fragment in 1% BSA PBS for 30 min on ice to block Fc receptors. Then, primary antibodies (biotinylated anti-LILRB2, anti-TREM2, or control IgG) were added at a final concentration of 10 μg/mL and incubated for 30 min on ice. After centrifugation to remove unbound primary antibodies, Alexa Fluor 488-labeled streptavidin (Jackson ImmunoResearch) was added to 2 μg/mL in 1% BSA PBS for 30 min on ice. After removal of unbound streptavidin by centrifugation, cells were analyzed using an iQue3 high throughput flow cytometer (Sartorius) with at least 10,000 live cells collected.

To prepare LILRB2-expressing cells, HEK293T cells were transfected with pcDNA3.1 carrying LILRB2 (aa1-598) with CMV-driven expression. After transfection for 72 hours, purified LILRB2 antibody (at designated concentration) in 1% BSA DPBS was incubated with transfected cells for 30 min on ice. Unbound antibody was removed by centrifuging cells at 350 rpm for 5 min. Then, Alexa Fluor 488-anti-human IgG (H + L) in 1% BSA DPBS (Thermo Fisher Scientific) at 2 μg/mL was incubated for 30 min on ice. After centrifuging cells at 350 rpm for 5 min to remove unbound secondary antibody, cells were analyzed using the iQue3 high throughput flow cytometer (Sartorius) with at least 10,000 live cells collected. The control cell line was transfected with a blank pcDNA3.1 plasmid.