Canine Endothelial Culture: A Case Study

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The purchased primary culture of the canine aortic endothelial cells (1 mL) will arrive frozen. The arrived sample will be thawed and put into the culture. While taking precautions, the ampule of canine aortic endothelial cells will be taken out of the liquid nitrogen storage and it will then be placed into the water bath (without submerging the cap) set 37◦C for about 1 to 2 minutes or as soon as ice melts. The ample of the mammalian cells will be wiped down with the paper towel moistened with 70% ethanol and 30% water. The minimum essential medium 1X (MEM) media will be pre-warmed to 37◦C in the water bath. Then, 11 mL of the MEM media will be transferred to a sterile T-75 flask through pipetting. The 1 mL of the canine’s cells will be pipetted…show more content…
The Reconstitution Buffer will be thawed and will be allowed to settle at room temperature along with the Lyophilized Luciferin Detection Reagent. At room temperature, the Reconstitution Buffer bottle will be completely transferred to the bottle containing Lyophilized Luciferin Detection Reagent and swirl to mix well. Approximately, 1 mL of the NAD/NADH-Glo assay will be performed. The reconstituted Luciferin Detection Reagent will be allowed to settle at room temperature. The Reductase will be thawed, Reductase Substrate and NAD cycling substrate will be centrifuged and placed on ice. About 275 uL of water will be added to the NAD cycling enzyme to mix and store the vial on ice. Then, 1 mL of NAD/NADH-Glo™ Detection Reagent is prepared by adding and mixing 1 mL of Reconstituted Luciferin Detection Reagent, 5 uL of Reductase, 5 uL of Reductase Substrate, 5 uL of NAD cycling enzyme and 5 uL of NAD cycling substrate. Unused reagents will be stored at -20◦C. The 96 well plate will be taken out of the incubator and allow to settle at room temperature for at least 5 minutes. Then, 100 uL of NAD/NADH-Glo™ Detection Reagent will be added to each of eight well to mix and lyse cells, so all the wells will have a total volume of 200 uL. The 96 well plate will be incubated at room temperature for 30 – 60 minutes. The luminescence will be recorded using a luminometer. The graph will be created to show the reduced metabolism mechanism of NAD/NADH in canine aortic endothelial cells exposed to arsenic and cadmium toxicity
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