Cyquant proliferation assay

3T3-L1 cells were obtained from ATCC. H460, Hela, BT549, and MCF7 cells were obtained from Washington University. All cells were found to be negative for mycoplasma contamination. All cells were cultured in high-glucose DMEM (Life Technologies) containing 10% fetal bovine serum (FBS) (Life Technologies) and 1% penicillin/streptomycin (Life Technologies) at 37°C with 5% CO₂. To establish a growth curve, cells were collected every 12–24 hr and counted in trypan blue with an automated cell counter (Nexcelom). For assessing proliferation, cells were grown under various experimental conditions for 48–72 hr, and proliferation was determined by manual cell counting or by using a CyQUANT proliferation assay (Thermo) according to the manufacturer’s instructions. For serum starvation, cells were cultured in DMEM (without FBS) for 48 hr. Proliferation was induced by transferring cells to media containing serum (20% FBS).

200,000 cells were handled and treated as described above for OCR assays. On the day of the assay, cells were washed with Seahorse XF RPMI supplemented with 1 mM glutamine. Glucose was left out of the medium so that basal rates of glycolysis could be measured upon stimulation with glucose at the start of the assay. Sodium pyruvate, a product of glycolysis that feeds into the TCA cycle and oxidative phosphorylation, was also not supplemented in the medium. ECARs were measured using a Seahorse XF24/XFe24 analyzer. Drug injections are provided in the order in which they occurred along with a brief description of their purpose as provided by the manufacturer's protocol below. First from Port A, glucose was injected to a final concentration of 10 mM to stimulate glycolysis. Next, from Port B, oligomycin was injected to a final concentration of 1 µM. As addition of oligomycin blocks ATP synthase, ATP production can only occur through glycolysis, resulting in increased ECAR values. Finally, from Port C, 2-deoxy-D-glucose is injected to a final concentration of 50 mM. 2-Deoxy-D-glucose is an analog of glucose that inhibits glycolysis, which results in decreased ECAR values. ECAR was normalized to cell number using the CyQuant Proliferation assay (Thermo Fisher Scientific, C7026).

Co-cultured cell proliferation was monitored using CyQUANT proliferation assay (Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer’s protocol. Briefly, following co-culture, the supernatant was removed, and cells were trypsinized and pelleted. The cell pellet was lysed by adding the lysis buffer containing the dye (provided in the kit). Fluorescence was measured in a microplate reader (Tecan, Switzerland) with excitation at 485 nm and emission detection at 530 nm.