DE-2–3 cells were cultured and differentiated essentially as described in Pan et al. (2009) (link) and Shen et al. (2017) (link). Briefly, cells were passaged in growth media consisting of high glucose DMEM supplemented with 10% fetal bovine serum, penicillin and streptomycin (100 U/mL), and GlutaMAX (2mM). For differentiation, cells were seeded at day −2 in differentiation medium (DM, growth media supplemented with 20nM insulin and 1nM T3) to reach confluence on day 0. On day 0, media was switched to induction media (DM supplemented with 0.5mM IBMX, 0.5 μM dexamethasone, and 0.125mM indomethacin). Media was changed to DM on day 2 and day 4. Cells were considered to be fully differentiated on day 6, and experiments were performed on day 6–9. For oxygen consumption measurements, DE-2–3 cells were differentiated in Seahorse XF24 Cell Culture Microplates, and oxygen consumption rates were measured using a Seahorse XF24 Extracellular Flux Analyzer instrument.
Xf24 extracellular flux analyzer instrument
Manufactured by Agilent Technologies
The XF24 Extracellular Flux Analyzer is a laboratory instrument designed to measure the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of cells in real-time. The instrument uses fluorescent sensors to detect changes in the levels of oxygen and pH in the cell culture medium, allowing researchers to assess cellular metabolism and mitochondrial function.
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Lab products found in correlation
4 protocols using xf24 extracellular flux analyzer instrument
1
Differentiation of DE-2-3 Cells
2
Differentiation of DE-2-3 Cells
3
Mitochondrial Oxygen Consumption Measurement
4
Measuring Cellular Metabolism Dynamics
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Cells were seeded on a 96‐well plate with a density of 5 × 104 cells/well and treated as indicated. Cells were then treated with ECAR reagents according to the manufacturer’s recommendations (Abcam). The ECAR measurements were taken at 5‐minute intervals for a total assay time of 120 minutes by a microplate reader system (PerkinElmer) using excitation and emission wavelengths of 380 and 615 nm, respectively. The OCR was measured in real time using the XF24 extracellular flux analyzer instrument (Seahorse Bioscience) as described previously.35 (link), 36 (link) Protein concentrations were used to normalize the results.
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