γ glutamyltransferase ggt activity colorimetric assay kit

Organoids cultured in hypoxia (3% and 1% oxygen) condition were used for erythropoietin
(EPO) excretion assessment after incubation for 1 h, 3 h, 6 h, 12 h, 24 h 36 h and 48 h.
Following incubation, cell culture supernatants were collected from 3% oxygen and 1%
oxygen cultured organoids and concentrated 4× using an Eppendorf Vacufuge for EPO
production measurement using a Human EPO ELISA kit (RayBiotech, Norcross, GA, USA)
according to the manufacturer’s instructions. EPO levels were determined via optical
density at 450 m wavelength absorbance. All samples were assayed in duplicate. A bar plot
was generated to illustrate EPO level.
Drugs excreted by the kidneys, aspirin, penicillin G+ and cisplatin were used for
nephrotoxicity testing. The organoids were treated with 200 μM aspirin, 200 μM penicillin
G and 200 μM cisplatin for 2 days in vitro. Drug-treated and normal renal
organoids, as well as 2D cultured renal cells were homogenized in 200 μl ice-cold GGT
Assay Buffer. The γ-Glutamyltransferase (GGT) Activity Colorimetric Assay Kit
(Sigma-Aldrich, St. Louis, MO, USA) was used according to the manufacturer’s protocol.

The purity of the depleted brain capillaries and parenchyma was analyzed by measuring the activity of γ-glutamyl transferase in the sampled fractions using the γ-Glutamyl Transferase (GGT) Activity Colorimetric Assay kit (Sigma-Aldrich, Brøndby, DK) according to the manufacturer's protocol. Briefly, sample volumes corresponding to a total of 10 mg tissue were taken from each fraction and homogenized in ice-cold GGT Assay buffer. The homogenized tissue samples were then centrifuged at 13,000 ×g for 10 min at 4 ºC to pellet any insoluble material. Ten microliters of the supernatant were transferred into a flat-bottom 96-well plate together with positive controls and standard curve samples. Additional 90 µL GGT Substrate Solution were added to each well (except for the standard curve samples), and the mixture was allowed to incubate for 3 min at 37 ºC before taking an initial measurement of the absorbance at 418 nm using an EnSpire Multimode Plate Reader (Perkin-Elmer, Waltham, MA, USA). The samples continued to incubate at 37 ºC and the absorbance at 418 nm was measured every 5 min to follow the conversion process. The initial and final absorbance measurements were used for calculating the enzyme activity presented as nmol/min/mL.