Cell-secreted TIMP-1 (DTM100, R&D Systems, Minneapolis, MN, USA), TIMP-2 (DTM200, R&D Systems, Minneapolis, MN, USA), MCP-1 (DCP00, R&D Systems, Minneapolis, MN, USA), and OPG (RAB0484, Sigma-Aldrich, St. Louis, Missouri, USA) were measured with the respective ELISA kits containing pre-coated ELISA plates, and the assays were performed as described by the manufacturers. Briefly, 100 μL of cell culture supernatant or standard was incubated in each well for 3 h at RT. Then, the HRP-conjugated antibody was added and incubated for 1 h at RT, followed by aspiration and three washes. Next, horseradish peroxidase was added and incubated for 1 h, followed by aspiration and washes. 3,3′,5,5′-Tetramethylbenzidine substrate was added to each well and incubated for 30 min in a dark chamber. Stop solution was added, and the absorbency of all ELISAs was read at 450 nm with a plate reader (Bio-Rad® Microplate Absorbance Reader, Bio-Rad Laboratories Inc., Hercules, CA, USA).
Dtm200
Manufactured by R&D Systems
Sourced in United States
The DTM200 is a laboratory instrument designed for the measurement of protein concentration. It utilizes the Bradford method, a spectrophotometric technique, to quantify the amount of protein present in a sample. The device provides accurate and reliable protein concentration data to support research and analytical activities.
Automatically generated - may contain errors
Lab products found in correlation
Dtm100, by R&D Systems (3 mentions)
Ab197747, by Abcam (2 mentions)
Microplate absorbance reader, by Bio-Rad (1 mentions)
Dcp00, by R&D Systems (1 mentions)
Mk101, by Takara Bio (1 mentions)
Dmp100, by R&D Systems (1 mentions)
Mmp200, by R&D Systems (1 mentions)
Dmp900, by R&D Systems (1 mentions)
Dy1750b, by R&D Systems (1 mentions)
7 protocols using dtm200
1
Quantifying Cell Secreted Proteins
2
TIMP-2 Secretion from Skin Cells
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To analyze the TIMP-2 secretion from KFs and PNFs, supernatant levels of TIMP-2 were assayed using a commercially available enzyme-linked immunosorbent assay (ELISA, DTM200, R&D Systems, Minneapolis, Minn.). KFs and PNFs were seeded into silicon chambers and grown to a density of 1.5 × 105 cells per chamber. Then the culture medium was changed, and the silicon chambers were incubated for 72 hours. To evaluate the effect of the treatment with TIMP-2, procollagen type I C-terminal peptide (PIP; MK101, TaKaRa, Shiga, Japan), TIMP-1, and MMP-1 (DTM100 and DMP100, R&D Systems) were assayed using commercially available ELISA. KFs were seeded into 6-well dishes and grown to a density of 1.5 × 105 cells per well. The medium was then changed to serum-free DMEM containing 50 μg/mL l
3
Biomarkers for Postoperative Renal Dysfunction
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Blood and urine samples were collected immediately after surgery. The collected samples were allowed to stand for 30 minutes, then centrifuged at 3000 rpm at 4 °C for 10 minutes, and the supernatant was stored at –80 °C.
The concentrations of urine neutrophil gelatinase-associated lipocalin (NGAL) (CSB-E09408h, Cusabio, Wuhan, China), urine tissue inhibitor of metalloproteinase-1 (TIMP-2) (DTM200, R&D Systems, Minneapolis, MN, USA), urine insulin-like growth factor-binding protein 7 (IGFBP7) (ARG81498, Arigo Biolaboratories, Shanghai, China), plasma sPD-1 (CSB-E13643h, Cusabio, Wuhan, China) and sPD-L1 (CSB-E13644h, Cusabio, Wuhan, China) were measured by commercial ELISA kits. The testing process was carried out in full accordance with the instructions, and the testers turned a blind eye to the clinical data, and the competent physician turned a blind eye to the biomarker test results.
The concentrations of urine neutrophil gelatinase-associated lipocalin (NGAL) (CSB-E09408h, Cusabio, Wuhan, China), urine tissue inhibitor of metalloproteinase-1 (TIMP-2) (DTM200, R&D Systems, Minneapolis, MN, USA), urine insulin-like growth factor-binding protein 7 (IGFBP7) (ARG81498, Arigo Biolaboratories, Shanghai, China), plasma sPD-1 (CSB-E13643h, Cusabio, Wuhan, China) and sPD-L1 (CSB-E13644h, Cusabio, Wuhan, China) were measured by commercial ELISA kits. The testing process was carried out in full accordance with the instructions, and the testers turned a blind eye to the clinical data, and the competent physician turned a blind eye to the biomarker test results.
4
Quantification of MMP-14, TIMP-2 and MMP-2
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MMP-14, TIMP-2 and total MMP-2 tissue levels were quantified using sandwich enzyme-linked immunosorbent assay (ELISA) kits. A standard curve was run in each assay; all samples and standards were measured in duplicate and findings averaged. The TIMP-2 assay procedure was done according to the manufacturer’s instructions (DTM200 and MMP200, R&D Systems). The MMP-14 assay procedure was carried out according to the manufacturer’s instructions with the incubation time of the samples with the antibody cocktail increasing to two hours (ab197747, Abcam).
5
Quantification of MMP and TIMP Levels
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MMP-14, TIMP-2, MMP-9, and TIMP-1 tissue levels were quantified using sandwich enzyme-linked immunosorbent assay (ELISA) kits. A standard curve was run in each assay; all samples and standards were measured in duplicate and findings were averaged. The TIMP-2, MMP-9, and TIMP-1 assay procedures were done according to the manufacturer's instructions (DTM200, DMP900, and DTM100, R&D Systems). The MMP-14 assay procedure was done according to the manufacturer's instructions with the samples in the antibody cocktail's incubation time increasing to two hours (ab197747, Abcam).
6
Biomarker Measurement Protocols for AKI Diagnosis
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All biomarkers were measured in our central laboratory by standard protocols in a technician-blinded manner. The levels of renal cell arrest biomarkers, urinary TIMP-2*IGFBP7 (u[TIMP-2]*[IGFBP7]), were measured by ELISA kits (TIMP-2: DTM200, R&D Systems; IGFBP7: DY1334-05, R&D Systems) according to the manufacturer’s instructions. The levels of renal cell injury and inflammation biomarkers, urinary KIM-1 (uKIM-1) and urinary IL-18 (uIL-18), were measured by ELISA kits (KIM-1: DY1750B, R&D Systems; IL-18: ELH-IL18, RayBiotech) on the manufacturer’s instructions. All biomarkers were measured in triplicate and the intra- and inter- assay variability ranged 2–6% and 5–9%. Urinary albumin was quantified by an automatic analyzer and expressed as the ratio to urinary creatinine (UACR). All urinary biomarkers were normalized to urinary creatinine. Baseline eGFR was estimated by the CKD-Epidemiology Collaboration Eq. [15 (link)]. Levels of biomarkers measured on the day of initial AKI clinical diagnosis were used for all analysis.
7
Serum and Urinary Biomarker Quantification
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Serum transgelin and urinary IGFBP-7 and TIMP-2 levels were measured in urine and serum samples taken from patients and control groups under strict aseptic conditions. All samples were processed and then stored at a temperature below 20° C. Serum transgelin levels were quantified using ELISA kits for transgelin from Lifespan BioSciences, Inc (catalog no: LS-F12693). Human IGFBP7 ELISA kits from Abcam was used to measure urinary IGFBP-7 levels (catalog no: ab229894). Quantikine ELSA Human TIMP-2 Immunoassays were used to measure urinary TIMP-2 levels (R &D systems, Catalog Number DTM200). All urinary biomarker levels were normalized by dividing by urine creatinine.
Transgelin, IGFBP-7, and TIMP-2 were chosen due to the availability, reasonable price, and simple methodology of respective ELISA techniques.
Transgelin, IGFBP-7, and TIMP-2 were chosen due to the availability, reasonable price, and simple methodology of respective ELISA techniques.
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