Cyquant cell proliferation kit

HCT 116 cells, with and without pre-treatment of 1μM AGI-5198, were grown in 5% CO₂ at 37°C and OCR and ECAR was measured using an XFe96 analyzer (Seahorse Bioscience, North Billerica, MA, USA). HCT116 cells were plated in XF96 cell culture plates, 2.0*10⁴IDH1^WT/R132H HCT116 cells or 1.75*10⁴IDH1^WT/WT cells (to account for a more rapid proliferation of IDH1^WT/WT HCT116 cells relative to IDH1^WT/R132H HCT116 cells ([53 (link)] and our own observation), were seeded in each well of 96-well assay plates and incubated for 48 h prior to conducting the assay. For determination of OCR, medium was changed to DMEM supplemented with 10 mM glucose, 2 mM glutamine and 1.5 mM pyruvate. For the glycolytic flux, medium was changed to DMEM supplemented with 2 mM L-glutamine and the concentration of glucose added to initiate glycolysis was 25 mM. Four independent replicates were conducted with 10 technical replicates per cell line. Data were expressed as pmol of O₂ per minute and normalized by cell number measured by the CyQUANT Cell proliferation kit (Invitrogen™), which is based on a fluorochrome binding to nucleic acids. Fluorescence was measured in a microplate luminometer (ClarioStar BMG Labtech, Cary, NC, USA) with excitation wavelength at 485 ± 10 nm and emission detection wavelength at 530 ± 12.5 nm.

Transfected cells were harvested after 48 hr and migration or invasion assays were carried out using polycarbonate filters (8 mm pore size; Corning, Amsterdam, The Netherlands). Cells in serum-free media were plated into the upper chamber and allowed to migrate along a Fetal Calf Serum (FCS; Invitrogen) concentration gradient for 24 hr. The number of cells migrating to the lower chamber was assessed using the CyQuant Cell Proliferation Kit (Invitrogen). For invasion experiments, the polycarbonate filters were coated in Matrigel (100 μg/cm²; BDBiosciences, Oxford, UK) 24 hr prior to the assay, and incubated at room temperature overnight to dry under sterile conditions. The Matrigel was rehydrated with serum-free media 30 min before the addition of cells. Cells were allowed to invade through this layer towards FCS for 72 hr prior to counting.

The oxygen consumption rate (OCR) was measured with the Seahorse XF96 extracellular flux analyzer (Agilent Technologies) as described previously (Shimura et al., 2019). Fibroblast cells were seeded in a 96‐well plate at 2 × 10⁴ cells/well with 80 μl of growth medium containing 25 mM glucose (Glu), and incubated for 24 h (37°C, 5% CO₂). After replacing the medium with 160 μl of unbuffered DMEM containing 1 mM sodium pyruvate, 2 mM glutamine, and 25 mM glucose or 10 mM galactose (Gal), the assay plates were incubated at 37°C without CO₂ for 1 h. Following the calibration of the sensor cartridge loaded with compounds including oligomycin (2 μM final concentration), carbonyl cyanide 4‐(trifluoromethoxy) phenylhydrazone (FCCP, 0.4 μM final concentration), and rotenone (1 μM final concentration), the experiments were started. The obtained data were normalized to the cell numbers determined using CyQUANT Cell Proliferation kit (Invitrogen). The data were calculated from two experiments performed with more than two replicates.

Oxygen consumption rate (OCR) was measured in PKAN and control neurons with a XF96 Extracellular Flux Analyzer (Seahorse Bioscience, Billerica, MA, USA). Each control and PKAN NPC was seeded on a XF 96‐well cell culture microplate (Seahorse Bioscience) at a density of 15–20 × 10³ cells/well and differentiated as previously described. After replacing the growth medium with 180 μl of bicarbonate‐free DMEM pre‐warmed at 37°C, cells were incubated at 37°C without CO₂ for 1 h before starting the assay procedure. Then, baseline measurements of OCR, after addition of 1 μM oligomycin and of 2.1 μM carbonyl cyanide 4‐(trifluoromethoxy) phenylhydrazone (FCCP), were measured using an already established protocol (Invernizzi et al, 2012). Data were expressed as pmol of O₂ per minute and normalized by cell number measured by the CyQUANT Cell proliferation kit (Invitrogen), which is based on a fluorochrome binding to nucleic acids. Fluorescence was measured in a microplate luminometer with excitation wavelength at 485 ± 10 nm and emission detection wavelength at 530 ± 12.5 nm. All measurements were performed in 24 replicates for each sample. At least three different experiments were carried out in different days. Experiments were carried out in blind conditions to the examiner.

Intracellular ATP levels were determined using the ATPlite Assay kit (Perkin Elmer) according to the slightly modified standard procedure. The method is based on the luciferin-luciferase reaction with ATP. Following ALA/SFC treatment, the medium in the 96-well plates was removed and the cells were washed with 200 µL of PBS. After adding 25 µL of mammalian cell lysis solution and 50 µL of PBS, the plates were shaken for 5 min at 700 rpm. Then, 37.5 µL of sample and 12.5 µL of substrate solution were added to the white-wall, clear-bottom 96-well plate, shaken for 5 min at 700 rpm, and incubated for 10 min in the dark. The luminescence levels were measured using a plate reader (Tecan Japan). ATP levels were corrected by the number of cells determined using the CyQUANT^® Cell Proliferation kit (Invitrogen) and evaluated in comparison with the corresponding fibroblasts without treatment.