Mouse monoclonal igg1 isotype control

For anti-CCL11 treatment, cancer cells were incubated with neutralizing human CCL11 monoclonal antibody or a mouse monoclonal IgG1 isotype control (R&D system, USA). Tumor cells were harvested at various time-points for analysis, each experiment was repeated five times.
For CCR3 gene silencing, custom-made plasmids carrying CCR3-shRNA were obtained (Anji Biotech, China). For transfection, cancer cells were cultured overnight at the logarithmic growth phase, then transfected with the studied vectors or negative control with Lipofectamine 2000 (Invitrogen, USA) according to the manufacturer's instructions for 72 hours. Stable transfected clones were validated by qRT-PCR and immunoblotting.

U937 cells were differentiated with 20 nM PMA or DMSO (control) at 5×10⁵ cells/ml/well for 24–72 h under normal cell culture conditions. At each time point 4 wells were washed once with cold PBS and dislodged with Accutase 200µl/well for 30 minutes. 5×10⁵cells/tube were washed twice with 500µl/tube cold Hepes buffer containing 3% BSA. Cells were blocked with 50µl buffer containing Human TruStain FcX (50µl/ml, Biolegend, San Diego, CA, USA) for 10 minutes at RT. After blocking, 50µl monoclonal anti PCI antibody (4PCI; Technoclone, Vienna, Austria) or mouse monoclonal IgG1 isotype control (R&D Systems, Minneapolis, MN, USA) were added to reach a final concentration of 30µg/ml and incubated for 1 h RT. Cells were washed twice and incubated with 50µl secondary Alexa Fluor 488 F(ab')2 fragment of goat anti-mouse IgG (H+L) (diluted 1∶1000, Invitrogen, Life Technologies, Carlsbad, CA, USA) for 45 minutes at RT. After washing, cells were fixed in 1% PFA and stored in the dark until FACS analysis. PCI detection was quantified by the mean fluorescent intensity multiplied with the percentage of Alexa 488 positive cells. The isotype control was substracted.