Igg2b isotype

BTRT cells were seeded in the growth medium. After attaching, cells were serum starved for 24 hours then FITC-BrdU pulse–labeled for 1 hour. After washing, cells were cultured in growth medium until collection. For testing the effects of treatment with docetaxel on CXCR4 expression, cells were treated with docetaxel (5nM) for 24 hours before BrdU pulse and after BrdU pulse until cell collection. BrdU was detected with FITC-conjugated anti-BrdU antibody. CXCR4 was detected with the anti-human CXCR4 antibody MAB172 (R&D, Minneapolis, MN), with the IgG₂B isotype (R&D) as a control, followed by staining with APC-conjugated secondary antibody (Invitrogen). Then cells were counterstained with 7-amino-actinomycin D (7-AAD) and analyzed using Beckman Coulter Gallios flow cytometer with Kaluza Analysis software.

BTRT cells were seeded in the growth medium. After attaching, cells were serum starved for 24 h then FITC-BrdU pulse-labeled for 1 h. After washing, cells were cultured in growth medium until collection. For testing the effects of treatment with docetaxel on CXCR4 expression, cells were treated with docetaxel (5 nM) for 24 h before BrdU pulse and after BrdU pulse until cell collection. BrdU was detected with FITC-conjugated anti-BrdU antibody. CXCR4 was detected with the anti-human CXCR4 antibody MAB172 (R&D, Minneapolis, MN), with the IgG₂B isotype (R&D) as a control, followed by staining with APC-conjugated secondary antibody (Invitrogen). Then cells were counterstained with 7-amino-actinomycin D (7-AAD) and analyzed using Beckman Coulter Gallios flow cytometer with Kaluza Analysis software.

Primary human neutrophil degranulation was assessed by measuring myeloperoxidase (MPO) secreted into the cell culture medium. Isolated neutrophils were resuspended in RPMI-1640 containing L-glutamine (ThermoFisher Scientific) and supplemented with 0.1% (w/v) fatty acid free BSA. 1x10^5 neutrophils were plated at a final volume of 100 μl in each well of a 96-well round bottom polypropylene plate (Corning Inc.) and pre-incubated with CCRL2 antibodies K097F7 (Biolegend), 152254 (R&D Systems) or isotype controls for 15 min. IgG2a isotype (Pfizer Inc.) and IgG2b isotype (R&D Systems) were used as controls for K097F7 and 152254, respectively. Neutrophils were then primed with 5 μg/ml cytochalasin B (Sigma-Aldrich, St. Louis, MO) for 15 min, followed by stimulation with 100 ng/ml of CXCL8 for 30 min. Plates were then centrifuged at 1000 rpm for 1 min and supernatants were collected. Supernatant MPO was quantified using the Human Myeloperoxidase (MPO) ELISA Kit (Mesoscale Discovery, Rockville, MD) following the manufacturer’s instructions. All incubation steps were performed at 37°C, 5% CO₂.