Cells were grown in 8-well culture miniplates (Seahorse XFp 103025-100, Agilent, Santa Clara, CA). The Cell Mito Stress Test (103 010–100, Agilent) was used to assess respiration. Seahorse XFp sensor cartridge ports were loaded with assay drugs dissolved in Seahorse base medium (103 334–100, Agilent) supplemented with 5 m m glucose, 1 m m pyruvate, and 4 m m glutamine. Oligomycin (1 µm ), FCCP (1 µm ), and Rotenone/Antimycin A (0.5 µm ) were used to modulate the respiratory chain (RC). After injection of each drug, OCR was measured in 3 cycles, each consisting of 3 min of mixing followed by 3 min of measurement. Confluent cell monolayers were undisturbed following completion of treatment TDF protocols. Thus, upon commencing the assay, numbers of cells in treated and untreated groups were all but identical.
Seahorse base medium
Manufactured by Agilent Technologies
Sourced in United States
Seahorse base medium is a cell culture medium designed to support the growth and metabolism of cells in the Seahorse XF Analyzer. The medium is optimized to maintain cell health and function during real-time measurements of cellular oxygen consumption rate (OCR) and extracellular acidification rate (ECAR).
Automatically generated - may contain errors
Lab products found in correlation
Cell mito stress test, by Agilent Technologies (1 mentions)
Xf24 cell plates, by Agilent Technologies (1 mentions)
Seahorse xfe96 analyzer, by Agilent Technologies (1 mentions)
Oligomycin, by Agilent Technologies (1 mentions)
Glut4, by Cell Signaling Technology (1 mentions)
P ampk, by Cell Signaling Technology (1 mentions)
Beclin 1, by Cell Signaling Technology (1 mentions)
P stat5, by Cell Signaling Technology (1 mentions)
P src, by Cell Signaling Technology (1 mentions)
Glut1, by Cell Signaling Technology (1 mentions)
Rotenone antimycin a, by Agilent Technologies (1 mentions)
P p70s6k thr389, by Cell Signaling Technology (1 mentions)
Carbonyl cyanide p trifluoromethoxyphenylhydrazone fccp, by Agilent Technologies (1 mentions)
P bcr abl, by Cell Signaling Technology (1 mentions)
P crkl, by Cell Signaling Technology (1 mentions)
Bcr abl, by Cell Signaling Technology (1 mentions)
Annexin 5 fitc pi apoptosis kit, by BD (1 mentions)
Cleaved parp, by Cell Signaling Technology (1 mentions)
P mtor, by Cell Signaling Technology (1 mentions)
P70s6k, by Cell Signaling Technology (1 mentions)
4 protocols using seahorse base medium
1
Cell Bioenergetics Profiling with Seahorse XFp
2
Metabolic Profiling of Oligodendrocytes
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Following papain dissociation, cells were plated onto XF24 cell plates (Seahorse Bioscience, Billerica, MA) pre-coated with matrigel, laminin and fibronectin (as above). Optimization of reagents was performed and the protocols and algorithm program were used according to the XF24 analyser. Briefly, cells were transitioned from their oligodendrocyte specific media into the Agilent Seahorse Base Medium which has low buffering capacity and 0 mM glucose and maintained in ambient CO2 for 25 min. The plate was then inserted into the machine; baseline oxygen consumption rate and extracellular acidification rate were measured prior to the addition of glucose (10 mM), oligomycin (1.5 μM) and 2-deoxy-glucose (100 mM). Both oxygen consumption rate and extracellular acidification rate were measured three times following the injection of each drug.
3
MitoStress and Glycolysis Assays
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For the MitoStress test, cells were seeded at 15,000 cells per well in an XF96 cell culture plate with the appropriate medium. The XF96 probes were calibrated with 200 µL of calibrant solution and incubated at 37 °C in the absence of CO2. After 12 h, the medium was changed to Seahorse base medium (Seahorse Biosciences, Santa Clara, CA, USA) supplemented with 10 mM glucose, 2 mM glutamine, and 1 mM sodium pyruvate and adjusted to pH 7.4. The probe cartridge was prepared to have 0.5 µM oligomycin in port “A”, 0.25 µM FCCP in port “B”, and 0.25 µM Rotenone with 0.25 µM Antimycin in port “C”. For the glycolysis stress test, the probe cartridge was prepared to have 20 µL of 50 mM glucose in port “A”, 11 mM oligomycin in port “B”, and 650 mM 2-deoxyglucose in port “C”. ECAR was normalized to mpH/min. Oxygen consumption rate and extracellular acidification rate were measured by the Seahorse XFe96 Analyzer (Seahorse Biosciences).
4
Investigating Imatinib Resistance in CML
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The human CML cell line KBM5 (harboring BCR-ABL) and its IM-resistant sub-line with T315I mutation (KBM5-T315I cells) were cultured at 37 °C with 5% CO2 in Iscove’s Modified Dulbecco’s Media (IMDM, from Gibco, USA) supplemented with 10% fetal bovine serum (FBS, from Hyclone, USA) as we described previously [9 (link)]. KBM5-T315I cells were routinely maintained in IMDM medium with IM (1.0 μM), which was removed 2–3 days before KBM5-T315I cells were used for experiments. IM was purchased from Selleckchem (Houston, TX, USA) and 2-DG was obtained from Cayman Chemical (Ann Arbor, MI, USA). Annexin V-FITC/PI apoptosis kit was from BD Biosciences (San Jose, CA, USA). Seahorse base medium, oligomycin, Carbonyl cyanide-p-trifluoromethoxy phenylhydrazone (FCCP), and rotenone/antimycin A were obtained from Seahorse Bioscience (North Billerica, MA, USA). Antibodies against AMPK(#5832), p-AMPK(#2537), Bcr/Abl(#3908), p-Bcr/Abl (#3901),beclin-1 (#4122), intact and cleaved caspase-8 (#9748, #9746), cleaved PARP (#5625), Glut1 (#12939), Glut4 (#2213), HK I (#2024), HK II (#2106), LC3A (#4599), mTOR (#4517), p-mTOR (#5536), p-p70S6K (Thr389) (#97596), p70S6K (#34475), p-CrkL (#3181), p-SRC (#2101), p-STAT5 (#4322), β-actin (#3700) were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibody to total OXPHOS cocktail (#110411) was purchased from Abcam (Cambridge, MA, USA).
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