Human il 6 elisa kit

The concentrations of interleukin-6 (IL-6) and interleukin-8 (IL-8) were conducted utilizing Human IL-6 ELISA Kit (ab178013; Abcam) and Human IL-8 ELISA Kit (ab214030; Abcam) according to the manufacturers’ instructions.

Human IL-6 ELISA Kit (ab178013), Interferon-gamma ELISA Kit (ab100538), Human TNF-alpha ELISA Kit (ab181421), and Human IL-8 ELISA Kit (ab108869) were purchased from Abcam (Cambridge, UK). Cytokine levels were determined according to the manufacturer’s protocol. Briefly, 50 µL of each sample and blank was placed in 96-well plates and incubated at room temperature for 1 h. Then, each well was washed 3 times with wash buffer and 50 μL of 1× biotinylated antibody for 30 min. TMB development solution was, then, added and incubated for 10 min. Then, 100 μL of stop solution was added, and absorbance was measured at 450 nm using an Infinite M200 Microplate Reader (Tecan Infinite M200 Pro; Tecan GmbH, Männedorf, Switzerland).

The measurement of TNF-α, IL-6, and IL-8 expression was performed using reagents provided in the Human TNF-α ELISA Kit (ab181421), Human IL-6 ELISA Kit (ab178013), and Human IL-8 ELISA Kit (ab214030) (Abcam, Cambridge, MA, USA), respectively. Furthermore, 2 × 10⁵/well keratinocytes were seeded in 24-well plate with or without 50 μg/mL of kinkéliba leaf extract treatment 24 h before use, with adherent cells collected by scrape after UVA irradiation and the subsequent treatment by kinkéliba leaf extract. The subsequent measurements were conducted according to the manufacturer’s instruction.

Concentrations of serum and HepG2 cells treated with serum drugs of IL1β, IL6, and TNFα were measured by ELISA kit (Abcam, United States, Mouse IL-6 ELISA Kit, ab222503), (Abcam, United States, Human IL-6 ELISA Kit, ab178013), (Abcam, United States, Mouse IL-1β ELISA Kit, ab197742), (Abcam, United States, Human IL-1β ELISA Kit, ab214025), (Abcam, United States, Human TNFα ELISA Kit, ab181421), (Abcam, United States, Mouse TNFα ELISA Kit, ab208348). The serum and cell samples were subjected to the 96-well plates and coupled with antibody cocktails after various treatments and co-incubated for 2 h. After another wash, the TMB Development Solution was incubated for 10 minis. The stop solution was added and the amount was measured by their corresponding calibration curves.

Peripheral venous blood samples were collected at admission, 3 days and 7 days. Serum levels of CRP, IL-1β, IL-6 and TNF-α was detected by ELISA using commercially kits: Human IL-1β ELISA Kit (ab46052), Human IL-6 ELISA Kit (ab229434), Human TNF-α ELISA Kit (ab229399) (all purchased from Abcam, Cambridge, MA, USA) and human CRP ELISA Kit (#MBS3800117, MyBioSource, Inc., San Diego, CA, USA).

A549 cells were plated into 24-well culture dishes at a cell density of 2 × 10⁵ cells per well for 1 day at 37 °C in a 5% CO₂ atmosphere. After 1 day, A549 cells were stimulated with poly-(I:C) (100 μg mL⁻¹) or vehicle (water) and incubated for 6 h at 37 °C in a 5% CO₂ atmosphere. Poly-(I:C)-stimulated A549 cells were treated with PEA-OXA (0.1, 1 and 10 µM), PEA (0.1, 1 and 10 μM) or vehicle (dimethyl sulfoxide or methanol, respectively) and incubated for the indicated time. Poly-(I:C)-stimulated A549 cells were also treated with a TRPV1 antagonist, IRTX (0.1 μM), or PPAR-α antagonist, GW6471 (1 µM), in the presence or absence of PEA-OXA (10 µM) or PEA (10 μM) and incubated for the indicated time. After 6 h, the supernatants were collected, and the amounts of produced IL-6 were measured by using a human IL-6 ELISA kit according to the manufacturer’s instructions (Abcam) and by using a reader Glomax^® Explorer (Promega, Milano, Italy). Data are expressed as picograms per milliliter of IL-6.