Apoptosis assay
Flow cytometry (FCM) was applied to detect the involvement of LILRA3 in
apoptosis of U937 cells. U937 cells (4×105cells/well)
harboring the null vector (pWSLV-02) and overexpression plasmid
(pWSLV-02-LILRA3) were plated in 6-well plates. After 48 hours of
culture, the cells were harvested and washed twice with PBS. The cells
were then stained using an annexin V-PE/7-amino-actinomycin D (7-AAD)
kit
(BD,
San Jose, USA) following the manufacturer’s protocols. Annexin V-PE was
used to detect phosphatidylserine externalization, a hallmark of early
apoptosis, and 7-AAD was used to label DNA fragments, a sign of cell
death. The Q4 area (annexin V-PE positive and 7-AAD negative) represents
early apoptotic cells, and Q2 (both annexin V-PE and 7-AAD positive)
represents late apoptosis.