In order to investigate the threshold number of oocytes for mass spectrometry detection, tubes of 1, 5 and 10 oocytes from 16-week-old mice were placed in 80% methanol and flash frozen in liquid nitrogen and stored in – 80 °C until processing. In another experiment, COCs from 3 week old mice were cultured for 2 h in bicarbonate buffered aMEM (Gibco) supplemented with 3 mg/ml BSA ± 250 μM 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR; Toronto Research Chemicals) (n = 3 biological replicates). Oocytes were denuded of their cumulus cells prior to sample extraction.
5 aminoimidazole 4 carboxamide 1 β d ribofuranoside aicar
Manufactured by LGC
Sourced in Canada
5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR) is a laboratory chemical compound. It is a synthetic nucleotide analog that mimics the structure and function of adenosine monophosphate (AMP).
Automatically generated - may contain errors
Lab products found in correlation
Aicar, by Merck Group (1 mentions)
Dmem high glucose glutamax, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (1 mentions)
2 mercaptoethanol, by Thermo Fisher Scientific (1 mentions)
Pi3k inhibitor ly294002, by Merck Group (1 mentions)
3 protocols using 5 aminoimidazole 4 carboxamide 1 β d ribofuranoside aicar
1
Oocyte Mass Spectrometry Detection
2
mESC Proliferation and Differentiation Assay
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D3 and R1/E mESCs lines
(ATTC, Manassas, USA) were cultured at a low density in Dulbecco’s
modified Eagle’s medium (DMEM-Glutamax) (high glucose) (Gibco,
Carlsbad, CA, USA) supplemented with 15% fetal bovine serum (FBS)
(Hyclone, Logan, UT, USA), 1% non-essential amino acids (Gibco, Carlsbad,
CA, USA), 0.1 mM 2-mercaptoethanol (Gibco, Carlsbad, CA, USA), 2 mMl -glutamine (Gibco, Carlsbad, CA, USA), 100 U/mL penicillin
(Gibco, Carlsbad, CA, USA), and 0.1 mg/mL streptomycin (Gibco, Carlsbad,
CA, USA). The culture was carried out at 37 °C under 5% CO2. Undifferentiated state was maintained by adding 1.000 U/mL
leukemia inhibitor factor (LIF) (Chemicon, Charlottesville, VA, USA)
to the culture medium. For experiments with mESCs, 150 cells/cm2 were cultured for 120 h in colonies in the absence or presence
of LIF and treated with 1 and 0.5 mM 5-aminoimidazole-4-carboxamide-1-β-d -ribofuranoside (AICAR) (Toronto Research Chemicals Inc., Ontario,
Canada), 2 mM metformin (METF) (Sigma, St. Louis, MO, USA), 2.5 μM
compound C (COMP C) (Sigma, St. Louis, MO, USA), and 20 μM PI3K
inhibitor LY294002 (Sigma) (Sigma, St. Louis, MO, USA) for 24, 72,
and 120 h; the inhibitor was added 30 min before AICAR treatment.
(ATTC, Manassas, USA) were cultured at a low density in Dulbecco’s
modified Eagle’s medium (DMEM-Glutamax) (high glucose) (Gibco,
Carlsbad, CA, USA) supplemented with 15% fetal bovine serum (FBS)
(Hyclone, Logan, UT, USA), 1% non-essential amino acids (Gibco, Carlsbad,
CA, USA), 0.1 mM 2-mercaptoethanol (Gibco, Carlsbad, CA, USA), 2 mM
(Gibco, Carlsbad, CA, USA), and 0.1 mg/mL streptomycin (Gibco, Carlsbad,
CA, USA). The culture was carried out at 37 °C under 5% CO2. Undifferentiated state was maintained by adding 1.000 U/mL
leukemia inhibitor factor (LIF) (Chemicon, Charlottesville, VA, USA)
to the culture medium. For experiments with mESCs, 150 cells/cm2 were cultured for 120 h in colonies in the absence or presence
of LIF and treated with 1 and 0.5 mM 5-aminoimidazole-4-carboxamide-1-β-
Canada), 2 mM metformin (METF) (Sigma, St. Louis, MO, USA), 2.5 μM
compound C (COMP C) (Sigma, St. Louis, MO, USA), and 20 μM PI3K
inhibitor LY294002 (Sigma) (Sigma, St. Louis, MO, USA) for 24, 72,
and 120 h; the inhibitor was added 30 min before AICAR treatment.
3
Quantifying Mitochondrial DNA Levels
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To obtain the relative mtDNA copy number, c2MPs or p107KO cells grown on a 6 cm tissue culture plate were untreated or treated with 1 mM 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR) (Toronto Research Chemicals) for 24 h in the presence of 5.5 mM or 25 mM glucose, with or without 10 mM Nam or 1μM srt1720 (detailed protocol in Supplemental Materials and Methods ).
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