Kinase experiment:

CHO-CXCR1 and CHO-CXCR2 membranes are prepared. Assays are performed in 96-well microtiter plates where the reaction mixture contained 1.0 μg/mL membrane protein in 20 mM Bis-Tris-propane, pH 8.0, with 1.2 mM MgSO4, 0.1 mM EDTA, 25 mM NaCl, and 0.03% CHAPS and SB 225002 (10 mM stock in Me2SO) added at the indicated concentrations, the final Me2SO concentration is<1% under standard binding conditions. Binding is initiated by addition of 0.25 nM 125I-IL-8 (2,200 Ci/mmol). After 1-h incubation at room temperature the plate is harvested using a Tomtec 96-well harvester onto a glass fiber filtermat blocked with 1% polyethyleneimine, 0.5% BSA and washed three times with 25 mM NaCl, 10 mM TrisoHCl, 1 mM MgSO4, 0.5 mMEDTA, 0.03% CHAPS, pH 7.4. The filter is dried, sealed in a sample bag containing 10 mL of Wallac 205 Betaplate liquid scintillation fluid, and counted with a Wallac 1205 Betaplate liquid scintillation counter[1].

Cell experiment:

Three esophageal squamous cell carcinoma cell lines WHCO1, WHCO5, and WHCO6 originally established from surgical biopsies of primary esophageal squamous cell carcinomas are cultured in DMEM containing 10% FCS at 37℃ in a humidified atmosphere of 5% CO2. MTT assays are carried out using the Cell Proliferation kit. Briefly, 1.5×103 cells are plated in 96-well plates in a final volume of 180 μL DMEM per well. SB 225002 (400 nM) is added to cells and 0.001% DMSO (solvent) is added as a control. After the indicated incubation period, 18 μL of the MTT labeling reagent (final concentration 0.5 mg/mL) is added to each well and incubated for 4 hours in a humidified atmosphere. One hundred eighty microliters of the solubilization solution are added to each well and the plates are left overnight at 37℃. The spectrophotometric absorbance of samples is measured at 595 nm using a microtiter plate reader[2].

Animal experiment:

Mice[3] Male 7-8 weeks old wildtype (C57BL/6J, Harlan) and ApoE–/– mice, which are generated on the same C57BL/6 background, are either fed with a normal chow or a cholesterol rich chow for 6 weeks and submitted to 20 min of left-sided middle cerebral artery occlusion (MCAO) or sham surgery. Animals are randomly attributed to treatment paradigms, and experimenters are blinded at all stages of interventions and data analysis. The selective CXCR2 antagonist SB225002 (2 mg/kg) or vehicle (1% DMSO in PBS) is injected intraperitoneally (i.p.) at 0, 24 and 48 hours post-ischemia. In other experiments, CXCR2 is specifically blocked by i.p. injection of a neutralizing rabbit anti-CXCR2 serum (300 μL) at 0 hours, 24 hours and 48 hours post-ischemia. In the latter studies, normal rabbit serum (NRS) served as control. In some experiments, neutrophils are depleted by i.p. injection of 200 μg anti-mouse Ly6G 24 hours before and 24 hours after ischemia. In these experiments, 200 μg of an isotype control antibody is delivered as control. Rats[4] In this study, 10-12 Sprague-Dawley rat pups per dam are used. The pups receive intraperitoneal injections of SB225002 (1 or 3 mg/kg, diluted in NS containing 0.33 % Tween 80) or vehicle (NS solution containing 0.33 % Tween 80) 30 min before lipopolysaccharide (LPS) administration and immediately after hypoxic ischemia (HI). The pups are randomly assigned to four groups: control (pups unexposed to LPS or HI, N=14), vehicle (NS injections 30 min before LPS administration and immediately after HI, N=18), and SB-1 (1 mg/kg, N=14) and SB-3 (3 mg/kg, N=18) (SB225002 injections 30 min before LPS administration and immediately after HI).

SB 225002 is a potent and selective CXCR2 chemokine receptor antagonist with IC50 value of 22nM [1].
CXCR2 is a member of the G-protein-coupled receptor family, which is responsible for neutrophil chemotaxis and margination induced by IL-8 [1].
SB 225002 is a potent and selective non-peptide inhibitor of CXCR2. It prevented IL-8 binding to CXCR2 with IC50 value of 22 nM and showed >150-fold selectivity over CXCR1. In HL60 cells, SB 225002 potently prevented IL-8 and GROa-induced neutrophil chemotaxis [1]. In CDDP-resistant and -sensitive OVCA cell lines, SB225002 induced apoptosis in both wild-type and p53-deficient cells in a p53-independent way and promoted mitotic catastrophe [2].
In rabbits, SB 225002 blocked IL-8-induced neutrophil margination [1]. In experimental colitis mice induced by TNBS, SB225002 reduced neutrophil influx, IL-1 , MIP-2, and keratinocyte-derived chemokine (KC) levels, MPO activity and the expression of vascular endothelial growth factor. While, levels of IL-4 and IL-10 were increased significantly in the colons of mice [3].
References:
[1]. White JR, Lee JM, Young PR, et al. Identification of a potent, selective non-peptide CXCR2 antagonist that inhibits interleukin-8-induced neutrophil migration. J Biol Chem, 1998, 273(17): 10095-10098.
[2]. Du M, Qiu Q, Gruslin A, et al. SB225002 promotes mitotic catastrophe in chemo-sensitive and -resistant ovarian cancer cells independent of p53 status in vitro. PLoS One, 2013, 8(1): e54572.
[3]. Manjavachi MN, Quintão NL, Campos MM, et al. The effects of the selective and non-peptide CXCR2 receptor antagonist SB225002 on acute and long-lasting models of nociception in mice. Eur J Pain, 2010, 14(1): 23-31.