Hyclone cosmic calf serum

Human colon epithelial cells, HCEC1CT and HCEC2CT cells were grown in DMEM media supplemented with Hyclone 199 media with EBSS (Thermo Fischer Scientific, MA), 2% Hyclone cosmic calf serum (Thermo Scientific, MA), EGF (20 ng/mL, Sigma-Aldrich, Milwaukee), hydrocortisone (1 μg/mL, Sigma-Aldrich, Milwaukee), insulin (10 μg/mL, Thermo Fischer Scientific, MA), apotransferin (2 μg/mL, Sigma-Aldrich, Milwaukee), sodium selenite (5 nM). HT29 cells were grown in McCoy’s 5A medium supplemented with 10% fetal bovine serum.

We measured cytotoxicity of the UGT inhibitors using a Pierce LDH Cytotoxicity Assay Kit (Thermo Scientific). HEK cells were seeded at 5 x 10⁴ per well in DMEM (Quality Biological) with 10% Hyclone Cosmic Calf Serum (Thermo Fischer), 200 μM of L-glutamine (Quality Biological), and 50 μg/mL of gentamicin (Quality Biological) at 37°C in 5% CO₂. We then incubated the cells with various concentrations of the UGT inhibitors overnight. Following this, we transferred 50 μL of media from each well to a new 96-well plate and then added 50 μL of reaction buffer. We incubated the mixture for 30 minutes and then added 50 μL of stop solution. We measured absorbance at 490 nm and 680 nm. We employed the following controls: a spontaneous LDH activity control which was incubated with the vehicle only and a maximum LDH activity control which was incubated with nothing but later lysed prior to incubation with the reaction buffer. We calculated absorbance for each well by subtracting the 680 nm absorbance value (background) from the 490 nm absorbance value. We then calculated percent cytotoxicity using the following equation:
$\%\text{Cytotoxicity}=\frac{\left(\text{UGT}\text{inhibitor}\text{LDH}\text{activity}-\text{Spontaneous}\text{LDH}\text{actvity}\right)}{\left(\text{Maximum}\text{LDH}\text{activity}-\text{Spontaneous}\text{LDH}\text{activity}\right)}$