Il 8 elisa kit

Cell-cultured medium was centrifuged, and the supernatant was subjected to ELISA detection according to the manufacturer’s instructions of the Human IL-6 ELISA Kit (BOSTER, #EK0410) and IL-8 Elisa Kit (BOSTER, #EK0413).
For tumor tissues, 0.1 g of tissue blocks were first transferred into a glass homogenizer containing 1 mL of pre-cooled PBS and 10 μL 100mM phenylmethylsulfonyl fluoride (PMSF), followed by thorough grinding and ultrasonication on ice. The prepared homogenate was centrifuged at 5000 × g for 5 min, and the supernatant was collected for ELISA assays.

The products of IL-6, IL-8 and TNF-α in the cell culture supernatants were examined by the human IL-6 ELISA kit (EK0410; Boster, Pleasanton, CA, USA), IL-8 ELISA kit (EK0413; Boster) and TNF-α ELISA kit (EK0525; Boster), respectively. Four paralleled wells were for each group. Following the assay protocols, OD absorbance was read with a microplate reader at 450 nm, and measured concentrations were determined using linear regression of OD value against the standard curve.

For the collection of conditional medium (CM), CAFs or NFs (1.25 × 10⁵ cells) were seeded in the collagen-coated flat bottom six-well plate and maintained in DMEM medium (Gibco) containing 1% or 10% FBS (Gibco) for 48 h. The medium was harvested and centrifuged at 800 × g for 5 min. The clear supernatant was then collected as CM.
For detection of the amount of secreted human IL-6, IL-8 in CAF-CM, or NF-CM, the enzyme-linked immunosorbent assay (ELISA) assay was performed using ELISA assay kit purchased from Boster Biological Technology: IL6 ELISA kit (Boster, Cat#EK0410), IL8 ELISA kit (Boster, Cat#EK0413). Dilution (1:10) was performed on the CM before quantifying the amount of cytokines according to the manufacturer’s protocol.