Enzyme linked immunosorbent assay kit

Permeabilized C2C12 cells undergoing calcium uptake capacity determination using CaGr as detailed above were collected at specified time points, and centrifuged at 21,000 g for 3 min. The supernatants were carefully removed, and both the supernatant and pellet fractions were immediately frozen and stored at −20 °C. Cytochrome c immunoreactivity was quantified in both fractions using an enzyme-linked immunosorbent assay kit (Abcam). Before measurement, the supernatant and pellet samples were diluted 1:20 and 1:200, respectively. The release of cytochrome c from in situ mitochondria is expressed as the content of cytochrome c in the supernatant as a percentage of the total content of cytochrome c present in the supernatant plus pellet.

Serum and urine creatinine, BUN, and serum cystatin C levels were measured using a colorimetric method with a Cobas 8000 analyzer (Roche, Mannheim, Germany). Urinary albumin level was measured using enzyme‐linked immunosorbent assay kit (Abcam, Cambridge, MA, USA). GFR was calculated using the equation (urine creatinine in mg/dl × urine flow rate in dl/hr)/serum creatinine in milligrams per deciliter.

Proteinase blocker (100 mg tissue/mL) was added to phosphate-buffered solution to help homogenise the HIP and PFC tissues. The mixture was then centrifuged at 12,000 rpm for 10 minutes. Tumor necrosis factor (TNF), interleukin-6 (IL-6), and interleukin-1 (IL-1) concentrations throughout the supernatant were measured in accordance with manufacturer recommendations using enzyme-linked immunosorbent assay kit (Abcam Pvt. Ltd., India) spectrophotometrically at 450 nm (model: UV-1800, Shimadzu, Japan). Standardized to total tissue weight, the cytokine concentrations in homogenates were represented as pg/100 mg tissue sample [62 (link)].

Quantitative measurement of rat C-reactive protein (CRP) present in liver tissue was performed using an Enzyme-Linked Immunosorbent Assay kit (Abcam^®). The provided CRP standard is used to prepare a standard curve to calculate the direct amount of the protein in samples. The minimum detectable amount of the protein for this assay is 0.7 ng/ml.
The homogenate was prepared as described above, in diluting buffer provided by the manufacturer (50 mg of liver tissue per 500 μl of buffer). The most suitable dilution of supernatant was determined empirically by performing a series of dilutions of pooled samples, in the range from 2 to 20 000. The 1:2000 dilution was chosen for this analysis since the detectable absorbance was within reader’s lower and upper limits. The assay was performed in accordance with the manufacturer’s protocol. Absorbance measurements were performed employing an Infinite^® 200 PRO microplate reader with i-control™ Software (Tecan Group Ltd, Männedorf, Switzerland). Each sample was analyzed in duplicate.

hASCs and its culture media components were purchased from Guangzhou Cyagen Biosciences co., LTD (Guangzhou, China). Penicillin–streptomycin, fetal bovine serum (FBS), Dulbecco's Phosphate Buffered Saline (D-PBS), and Trypsin were purchased from Gibco (USA). CD105-PE, CD73-PE, HLA-ABC-PE, CD14-FITC, CD34-PE, and CD45-PE antibody were purchased from Becton–Dickinson (USA). Trizol reagent was purchased from Life Technologies (USA). MiniBEST Universal RNA Extraction Kit, SYBR Premix Ex Taq II (Tli RNaseH Plus) kit, and PrimeScript RT Master Mix kit (Perfect Real Time) were purchased from TaKaRa (Japan). RNeasy Mini Kit was purchased from Qiagen (Germany). Cell cycle kit and Annexin V-FITC/PI apoptosis kit were purchased from Multisciences (Lianke) Biotech, co., LTD (Hangzhou, China). Cell Counting Kit-8 (CCK-8) kits were purchased from Dojindo (Osaka, Japan). Ki-67 cell proliferation kit (IF) was purchased from Sangon Biotech (Shanghai) Co., Ltd (Shanghai, China). Enzyme-Linked Immunosorbent assay kit was purchased from Abcam (United Kingdom). The Scepter 2.0 cell counter was purchased from Merck Millipore (Massachusetts, USA). The chromatographic column was purchased from Waters (USA). Acetonitrile and methanol were purchased from Merck (Darmstadt, Germany). Formic acid was purchased from CNW Technologies (Shanghai, China).