Infinite m1000 instrument

For cell proliferation assay, 2 × 10⁴ cells per well were seeded in a 96-well plate. After SW480 cell overexpression and HT-29 cell interference, the number was detected with Cell Counting Kit-8 (CCK8) (Beyotime, Shanghai, China) for 5 consecutive days. The absorbance at 450 nm was measured with Infinite M1000 instrument (Tecan Austria GmbH, Grödig, Austria).

The different BM BC cells (2 × 10³ cells/well) were seeded into 96-well plates for CCK8 assay and cultured at 37 °C in 5% CO₂, and siSNORA71B and siNC were transfected into the high BM BC cells with Lipofectamine 2000, respectively. In addition, pcDNA3.1-SNORA71B and pcDNA3.1 were, respectively, transiently transfected into the low BM BC cells. After 2 days, the cells were incubated with 10 μl of CCK8 reagent (Beyotime, Shanghai, China) at 37 °C for 4 h at the indicated time point for 0 h, 24 h, 48 h, 72 h, and 96 h. The absorbance at 450 nm was measured with Infinite M1000 instrument (Tecan Austria GmbH, Grödig, Austria). Each experiment was performed in triplicate.

Hepatoma HepG2 cells (200,000 cells/well of 12-well plates) were pretreated with DMSO (vehicle, 0.1% v/v) or EPA and/or DHA (10, 25, or 50 μM) for 3, 6, 16, or 24 H and then cultured for up to 24 H in the presence of a BA mixture (CDCA, CA, DCA, and LCA, 100 μM each). For FACS analyses, cells were washed with PBS and resuspended in 100 μL of binding buffer containing 5 μL of Annexin V-FITC and 10 μL of propidium iodine (PI) and incubated in the dark for 15 min. Labeled samples were then analyzed for Annexin V-FITC (525/50 nm) and PI (585/42 nm) using a FACSCanto II instrument (San Jose, CA, USA), at the flow cytometry platform in the CHU de Québec Research Centre (http://services.crchudequebec.ca/services/cytometrie/).
The caspase-3 activity was determined using the EnzChek caspase-3 Assay Kit according to the manufacturer's instructions (Thermo-Life Technologies). Results were analyzed with an Infinite M1000 instrument (Tecan, Austria).