E el m0044c

Renal tissues levels of mouse IL-17A, IFN-γ (MULTISCIENCES: EK217/2-AW1, EK280/3-AW1), IL-6, IL-4 and IL-10 (Elabscience: E-EL-M0044c, E-EL-M0043c, E-EL-M0046c) were quantified by using an ELISA method according to the instructions. Renal tissue lysates were extracted according to the following formula: 50 mmol/L Tris–HCl, 0.2% Triton X-100, 2 mmol/L EDTA, 150 mol/LNaCl, 2 mmol/L EGTA, 0.3%IGEPAL, 10 μl/ml proteinase inhibitors cocktail, 10 μl/ml PMSF, and 10 μl/ml orthovanadate (21 (link)).

ELISA analysis was performed as previously described (Yan et al., 2021 (link)). The inflammatory cytokines TNF-α, IL-6 and IL-1β in brain tissue or cell culture medium were detected with ELISA kits (Elabscience Biotechnology, E-EL-M0037c for mouse IL-1β, E-EL-H0149c for human IL-1β, E-EL-M0049c for mouse TNF-α, E-EL-H0109c for human TNF-α, E-EL-M0044c for mouse IL-6, E-EL-H0102c for human IL-6). All procedures were performed according to the manufacturer’s instructions.

Blood was collected from mice under anaesthesia via the ophthalmic vein into EDTA tubes and then centrifuged at 1,500 rpm for 15 min at 4°C. Serum samples were then divided evenly into the Eppendorf (EP) tubes and preserved at −80°C for subsequent experiments. TNF-α (Elabscience, China, #E-EL-M3063), IL-1β (Elabscience, China, #E-EL-M0037c), and IL-6 (Elabscience, China, #E-EL-M0044c) in serum were assayed with ELISA kits.

Articular cartilage tissue (50–100 mg) was obtained from each KOA patient during artificial joint replacement surgery. Total RNA was extracted using Qiagen 74106 tissue RNA extraction kit (Qiagen, Germany). The mRNA was reverse-transcribed into cDNA using the M-MLV2 reverse transcription kit. RT-PCR was performed in 25 μl reaction volumes containing 2 μl of cDNA, 12.5 μl of 2× PCR buffer, 15 mmol/l MgCl₂, 25 μmol/l each of the four dNTPs, 50 pmol/l primer, and 1 U of Taq enzyme. The primers used for RT-PCR were as follows: IL-6, 5′-GAG CTT CAG GCA GGC AGT ATC-3′ (forward) and 5′-GTA TAG ATT CTT TCC TTT GAG GC-3′ (reverse); IL-8, 5′-AGT GCT AAA GAA CTT AGA TG-3′ (forward) and 5′-TAT GAA TTC TCA GCC CTC TT-3′ (reverse); MMP-13, 5′-GGT CCC AAA CGA ACT TAA CTT ACA-3′ (forward) and 5′-CCT TGA ACG TCA TCA TCA GGA AGC-3′ (reverse); and β-actin, 5′-ACC ACC ATG GAG AAG GCT GG-3′ (forward) and 5′-CTC AGT GTA GCC CAG GAT GC-3′ (reverse). Synovial fluid (1 ml) from each KOA patient was collected and centrifuged at 14000 r/min for 20 min at 4°C, after which the supernatant was isolated for determination of IL-6 (Cat#E-EL-M0044c, Elabscience), IL-8 (Cat# E-EL-H0048c, Elabscience), and MMP-13 (Cat# E-EL-H0134c, Elabscience) levels by ELISA. All procedures were carried out in strict accordance with the manufacturers’ instructions.

At the end of the experiment, blood was obtained from the mice and their serum was collected following centrifugation at 3000 rpm for 30 min. The ELISA kits for measuring IL‐1β (E‐EL‐M0037c), IL‐6 (E‐EL‐M0044c), TNF‐α (E‐EL‐M3063), IL‐10 (E‐EL‐M0046c) and IL‐4 (E‐EL‐M0043c) were purchased from Elabscience (Wuhan, China). The levels of IL‐1β, IL‐6, TNF‐α, IL‐10 and IL‐4 in the mouse serum were quantified using the respective ELISA kits according to the manufacturer's instructions. The absorbance was determined on a microplate reader at OD450 nm.

Samples of mouse blood serum and the supernatants of HPAEpiCs were collected for analysis of cytokines, including Interleukin 1 Beta (IL-1β), Interleukin 6 (IL-6), Interleukin 17 (IL-17), and monocyte chemotactic protein 1 (MCP-1) by using Mouse IL-1β (E-EL-M0037c, Elabscience, China), IL-6 (E-EL-M0044c, Elabscience), IL-17 (3020092, BioAim Scientific Inc., Toronto, Canada), and MCP-1 (E-EL-M3001, Elabscience) ELISA kits according to the manufacturer's instructions.