Il 8 human uncoated elisa kit

Secretion of cytokines IL-8, TNF-α, and IL-10 into the basolateral compartment by the mammalian cells was measured with an enzyme-linked immunosorbent assay (ELISA) prior to inoculation and at t = 0, 2, 24, 48, and 72 h after inoculation. IL-8 was measured with a human IL-8 (CXCL8) Mini-TMB ELISA development kit (PeproTech) for the dual coculture assay, with a human IL-8 (CXCL8) standard ABTS ELISA development kit (PeproTech) for the triple coculture assay, and with a human IL-8 uncoated ELISA kit (Invitrogen) for the additional triple coculture assay with L. sakei and S. aureus, all performed according to the manufacturer’s instructions. IL-10 and TNF-α were measured with a human IL-10 Mini-ABTS ELISA development kit (PeproTech) according to the manufacturer’s instructions. Color development was measured with an Infinite M200 Pro plate reader (Tecan) at 405 nm with wavelength correction at 650 nm for ABTS substrate and at 450 nm with wavelength correction at 620 nm for TMB substrate.

ATRA-induced differentiated HL-60 cells were suspended in RPMI-1640 medium containing 10% fetal clone III, 4 mM L-glutamine, 100 units/mL penicillin, and 50 μg/mL streptomycin at a density of 2 × 10⁶ cells/mL. Human neutrophils were suspended in RPMI-1640 medium containing 10% FBS, 2 mM L-glutamine, 100 units/mL penicillin, and 50 μg/mL streptomycin at a density of 5 × 10⁶ cells/mL. Aliquots of 50 μL of suspension cells were added into individual wells of 96-well plates, and then 50 μL of D-PBS, pH 7.2, containing various stimuli, including HP-NAP, Pam₃CSK₄, and LPS, was added to each well. The final concentration of HP-NAP was 1 μM unless otherwise specified. The cells were incubated at 37 °C for 16 h. The cell suspensions were centrifuged at 600× g for 5 min. The amount of IL-8 present in the supernatant was measured by using the human IL-8 ELISA Ready-SET-Go kit (Cat. # 88-8086-88, eBioscience) or the human IL-8 uncoated ELISA kit (Cat. # 88-8086-88, Invitrogen, Vienna, Austria) according to the manufacturers’ instructions.

The antibodies used in the current study are listed in Table S1. The other reagents used are as follows: recombinant EGF, IL‐1β, IL‐7, IL‐13, IL‐15, GRO‐α, IL‐8, SDF‐1α, MCP‐3, MIP‐1α, MCP‐2, MIP‐3α, eotaxin‐3, bFGF, KGF, vEGF, IGF‐BP7, OPG, GM‐CSF (BBI Life Sciences, Shanghai, China); recombinant IL‐6, MMP‐3, TGF‐β1 (PeproTech); cetuximab (Merck KGaA); gefitinib, cisplatin, and LY3214996 (Selleck Chemicals Inc); MTT (3‐[4]‐2,5‐ diphenyltetrazolium bromide thiazolyl blue), X‐Gal (5‐bromo‐4‐chloro‐3‐indolyl‐β‐D‐galactopyranoside), DAPI (4′, 6‐diamidine‐2′‐phenylindole dihydrochloride), BrdU (5‐bromo‐2‐deoxyuridine), and PI (propidium iodide) (Sigma‐Aldrich); annexin V‐FITC/PI Apoptosis Detection Kit (Yeasen Biotech); Human IL‐8 Uncoated ELISA Kit (Thermo Scientific); Human Total MMP3 ELISA Kit and Human EGF ELISA Kit (Proteintech).