Reparixin

Human bronchial epithelial cells (16HBE, HBE135-E6E7) were obtained from Fuhen Biologicals. The cells were cultured in KM medium (Sciencell, Cat#2101) supplemented with 1% keratinocyte growth supplement (KGS, Cat. No. 2152) and 1% penicillin-streptomycin (P/S, Cat. No. 0503). They were maintained in a humidified cell culture incubator at 37 °C with 5% CO₂. To induce cell injury, cells were treated with LPS (Sigma, Cat: L2630) at concentrations ranging from 1 to 800 μg/mL for 24 h. Moreover, cells were treated with various concentrations of the CXCL1/CXCL2 inhibitor-Reparixin (0-40 μM, Selleck, Cat#S8640) for 24 h.
The CCK-8 assay is a widely used method for assessing cell proliferation and cytotoxicity. To perform the assay, 1×10^4 cells were plated in each well of a 96-well plate and then incubated for 24 h. Subsequently, LPS or Reparixin was added to six replicate wells in each group and the process was repeated 3 times. After 24 h of incubation, each well was treated with 10 μL of CCK-8 solution for 2 h (Abbkine, Cat#: BMU106-CN) and the optical density was measured at 450 nm.
After aspirating the culture solution from each well, we added 1 mL of Trizol to the cells in each well of the six well plate. The specific procedures and analytical methods were described above. The primer sequences were shown in Supplementary Table 3.

Neutrophils were isolated using the EasySep Neutrophil Isolation Kit (Stemcell Technologies), counted, and resuspended in RPMI containing L-glutamine and 5% FCS. Neutrophils were exposed to plasma (10% final concentration) derived from healthy donors (n = 4) or patients with COVID-19 (n = 7) and subsequently stained for indicated activation markers. For selected subgroups, human IL-8 (MilliporeSigma, I1654), anti-human IL-8 antibody (MilliporeSigma, I2519), or reparixin (20 μM, SelleckChem, S8640) were added to either plasma or neutrophils 20 minutes before neutrophil exposure to plasma. Cells were incubated at 37°C and 5% CO₂ for 1 hour, fixated using 1% PFA. Mean fluorescent intensities (MFIs) of neutrophils (singlets>size>CD15⁺+CD16⁺) were assessed.
In a separate series of experiments, isolated healthy neutrophils were added to poly-L lysine coated Ibidi μ-slides and treated with plasma (20% final concentration) and/or inhibitors as described above. Neutrophil granules, released vesicles and NETS were stained using antibodies against myeloperoxidase (MPO, R&D Systems, AF3667) and neutrophil alkaline phosphatase (ALPL, MilliporeSigma, HPA008765) and secondary antibodies (anti-goat AF594, anti-rabbit AF647, 1:200, Invitrogen) along with 4′,6-diamidin-2-phenylindole (1:1000) and SytoxGreen (Thermo Fisher Scientific, S7020, 500 nM final concentration, see Supplemental Figure 4C).

BGC823, SGC7901 and GES-1 cells were purchased from Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). All the cell lines were supplemented with 100 μg/ml streptomycin, 100 U/ml penicillin and 10% fetal bovine serum (FBS) at 37 °C in a humidified atmosphere of 5% CO₂. 5-fluorouracil, oxaliplatin, paclitaxel and reparixin (Selleck Chemicals, USA) were used at the indicated concentrations.