Recombinant cxcl1

AGS cells were treated with H. pylori, DAC, or TNFα and 24 h post-treatment supernatant was collected and stored at −80 °C. CXCL1 secretion in the supernatant from the treated cells was analyzed by using the CXCL1 ELISA kit (R&D System, Minneapolis, MN, USA), according to the manufacturer’s instructions. A standard curve using recombinant CXCL1 (R&D System, Minneapolis, MN, USA) was employed to determine CXCL1 concentrations in the control and treated samples and the values were presented in pg/mL.

Recombinant CXCL1 was purchased from R&D Systems Inc. Reparixin was purchased from American Custom Chemicals Corporation. CXCR-blocking antibodies reported previously69 (link) from Abcam were used at 2 μg ml⁻¹. CXCL1 concentration was determined by ELISA according to the manufacturer's protocol (R&D Systems Inc). All assays were performed independently three times. Neutralizing mouse monoclonal CXCL1 and CXCL8 antibodies (R&D Systems) were used at 1:1,000.

Pharmaceutical grade bleomycin was obtained from the University of Michigan Hospital and diluted in sterile PBS. Bleomycin was administered at a concentration of approximately 0.016 U per 20-gram mouse.
MRSA strain USA300 was obtained from the Network of Antimicrobial Resistance in Staphylococcus aureus. To prepare bacterial cultures for infections, MRSA was grown overnight at 37°C with constant shaking in Nutrient Broth (BD Difco). CFU were determined by optical density relative to a standard curve for an inoculation concentration of approximately 1 × 10⁷ or 7 × 10⁷ CFU per mouse. Experiments were terminated 24 hours postinfection unless otherwise specified in figure legends.
For experiments where opsonized MRSA was used, bacteria were resuspended to a concentration of 1 × 10⁸ CFU/mL and treated with 1 μL of 4 mg/mL anti–S. aureus polyclonal antibody (catalog ab20920) (Abcam) per 3 mL of bacterial culture. Cultures were rocked at 37°C for 30 minutes and then used for subsequent ex vivo infections.
Recombinant CXCL1 (R&D Systems, Bio-Techne) was administered to mice at a concentration of 1 μg/mouse.
LPS from Pseudomonas aeruginosa (MilliporeSigma) was administered to mice at a concentration of 25 μg/mouse as previously described (52 (link)).