Recombinant human rhesus macaque feline cxcl12 sdf 1 alpha

In the experiments with recombinant CXCL12, 1 × 10⁵ cells of parental HEK293T or UE7T-9 cells were seeded in a 24-well plate. After 48 h of incubation, 1 × 10⁵ cells of U937 were added to 1000 μl of fresh RPMI medium with or without recombinant Human/Rhesus Macaque/Feline CXCL12/SDF-1 alpha (1000, 10,000, 100,000 pg/μL, R&D Systems). U937 mono-culture groups with recombinant CXCL12 were also conducted in the experiment. After 48 h of co-culture or mono-culture, cells were exposed to 5 μM of cytarabine for 48 h. After 48 h of cytarabine exposure, all the cells in each well were collected, fixed with 4% paraformaldehyde, and permeabilized with 90% methanol following the manufacturer’s protocol. All collected cells were incubated for 1 h with anti-cleaved caspase-3 primary antibodies (1:6400) at room temperature, washed twice with PBS, and incubated with anti-rabbit IgG (H + L), F(ab’)2 Fragment (PE conjugate) for 30 min at room temperature and protected from light. Then, they were washed twice with PBS. The percentages of cleaved caspase-3-positive cells were evaluated using the BD FACSCanto II. The cells were gated based on PE-A and SSC-A channels as populations that exhibited negligible signals in the unstained negative controls.