ATP and ADP concentrations were measured by means of ATP Bioluminescence Assay Kit HS II (Roche Applied Science, Monza, Italy) and the ADP Colorimetric/Fluorometric Assay Kit (Sigma-Aldrich). Cells were harvested, counted, and lysed with the above-mentioned lysis buffer. Aliquots (50 µL) from each diluted sample or standard were transferred to 96-well plates, after which 50 µL of a luciferase reagent was added. After mixing, the emitted light was measured and integrated over 10 s on an automated microplate luminometer (Bio-Tek, San Diego, CA, USA).
Adp colorimetric fluorometric assay kit
Manufactured by Merck Group
Sourced in Italy, United States
The ADP Colorimetric/Fluorometric Assay Kit is a laboratory product manufactured by Merck Group. It is designed to detect and quantify the presence of ADP (adenosine diphosphate) in samples. The kit utilizes a colorimetric or fluorometric detection method to measure ADP levels.
Automatically generated - may contain errors
Lab products found in correlation
Human thrombin, by Merck Group (2 mentions)
Sensolyte 520 thrombin activity assay kit, by AnaSpec (2 mentions)
Thromboxane b2 txb2 elisa kit, by Enzo Life Sciences (2 mentions)
Infinite m200 reader, by Tecan (2 mentions)
6 phosphogluconate assay kit, by Abcam (2 mentions)
Atp colorimetric fluorometric assay kit, by Merck Group (2 mentions)
Pyruvate assay kit, by Merck Group (2 mentions)
Pep colorimetric fluorometric assay kit, by Merck Group (2 mentions)
Atp bioluminescence assay kit hs 2, by Roche (1 mentions)
Automated microplate luminometer, by Agilent Technologies (1 mentions)
6 protocols using adp colorimetric fluorometric assay kit
1
ATP and ADP Quantification Assay
2
Platelet Activation and Aggregation Assays
3
Platelet Activation and Aggregation Assays
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Platelets, platelet membrane vesicles, and PNPs of equivalent membrane content were prepared and examined for platelet-activating molecules, including thrombin, ADP, and thromboxane, using a SensoLyte 520 Thrombin Activity Assay Kit (AnaSpec), ADP Colorimetric/Fluorometric Assay Kit (Sigma Aldrich), and Thromboxane B2 (TXB2) ELISA Kit (Enzo Life Sciences), respectively, based on the manufacturers’ instructions. Each sample was assayed in replicate (n=3).
Aggregation of platelets in the presence of PNPs was assessed using a spectrophotometric method. 1 mL aliquot of platelet rich plasma (PRP) was first prepared from human whole blood with sodium citrate as the anti-coagulant. The plasma was then loaded into a cuvette followed by addition of 500 µL of 2 mg mL−1 PNPs in PBS solution. As negative and positive controls, the PRP was mixed with 500 µL of PBS or 500 µL of PBS containing 0.5 IU mL−1 of human thrombin (Sigma Aldrich), respectively. The cuvettes were immediately placed in a TeCan Infinite M200 reader and monitored for change in absorbance at 650 nm over time, and platelet aggregation was observed based on the reduction of turbidity.
Aggregation of platelets in the presence of PNPs was assessed using a spectrophotometric method. 1 mL aliquot of platelet rich plasma (PRP) was first prepared from human whole blood with sodium citrate as the anti-coagulant. The plasma was then loaded into a cuvette followed by addition of 500 µL of 2 mg mL−1 PNPs in PBS solution. As negative and positive controls, the PRP was mixed with 500 µL of PBS or 500 µL of PBS containing 0.5 IU mL−1 of human thrombin (Sigma Aldrich), respectively. The cuvettes were immediately placed in a TeCan Infinite M200 reader and monitored for change in absorbance at 650 nm over time, and platelet aggregation was observed based on the reduction of turbidity.
4
Quantification of Metabolites in Kidney AKI
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The amount of pyruvate was measured using Pyruvate Assay Kit (Sigma). Kidneys harvested from Akr1a1+/+, Akr1a1−/−, Pkm2+/+ or Pkm2−/− mice (sham operation or AKI) were mechanically homogenized in Pyruvate Assay Buffer (1 mg/5 μl buffer). After extracts were clarified by centrifugation (20,000g, 4 °C, 20 min, ×2), supernatant was used for assay. GHB in the serum of Akr1a1+/+ and Akr1a1−/− mice was measured following the GHB enzymatic assay kit from BUHLMANN. For measuring PEP, 6PG, ATP, ADP and serine in HEK cells, 1 × 106 cells were lysed in corresponding buffer. The amount of PEP, 6PG, ATP, ADP and serine were respectively measured using PEP Colorimetric/Fluorometric Assay Kit (Sigma), 6 Phosphogluconate Assay kit (abcam), ATP Colorimetric/Fluorometric Assay Kit (Sigma), ADP Colorimetric/Fluorometric Assay Kit (Sigma) and DL-Serine Assay kit (Fluorometric) (Biovision).
5
Enzymatic Kinase Activity Assay
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The reaction mixture contained 2 mM DMA as the substrate, 2 mM ATP as a coenzyme factor, 50 mM citrate buffer or Tris-HCl buffer, 2 mM MgCl2, and purified recombinant protein (40 μg) in a total volume of 100 μL. The reaction mixture was incubated at a suitable temperature for 20 min and terminated by boiling for 3 min. The samples were used to analyze the ADP concentration in order to calculate the production of DMAP. The concentration of ADP was determined using the ADP Colorimetric/Fluorometric Assay Kit (Sigma-Aldrich, St. Louis, MO, USA). The control was prepared according to the reaction without the recombinant promiscuous kinases.
6
Quantification of Metabolites in Kidney AKI
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