Human cell line BG02 and H9 embryonic stem cells maintained on MEFs as previously described54 (link). To induce cortical neuroepthelial stem cells, the ESCs were digested into small clumps for suspension culture on low-cell-adhesion dishes in the medium containing Advance DMEM/F12 (Gibco): Neurobasal media (Gibco) (1:1), 1×B27 (Gibco), and 1% Glutmax (Sigma), 10 ng/mL bFGF (Gibco), 3 μM CHIR99021 (StemRD), 5 μM SB431542 (Cellagen technology), 0.2 μM Compound E (Calbiochem), 0.1 μM LDN193189 (Selleck), and 50 μg/ml Vitamin C (Vc, Sigma). After 6 days, EBs were transferred to 5 μg/ml laminin (Gibco) and poly-ornithine (Sigma, 10 μl/well)-coated plates for attachment culture and the media was switched to CHbFSB + LIF (NESC) culture medium35 (link)36 (link). The NESC culture medium is composed of Neurobasal media surplus with 1×B27, 1×N2, 1XNEAA (Sigma), 1% Glutmax (Sigma), 3 μM CHIR99021, 5 μM SB431542, 10 ng/ml bFGF, and 1000 U/ml hLIF (Millipore).
Glutmax
Manufactured by Merck Group
Sourced in United States
Glutmax is a laboratory instrument designed for the measurement of glutamine, a common amino acid, in various biological samples. The device utilizes an enzymatic assay method to quantify the concentration of glutamine present in the sample. Glutmax is intended for use in research and analytical laboratory settings.
Automatically generated - may contain errors
Lab products found in correlation
Basic fibroblast growth factor (bfgf), by Merck Group (3 mentions)
Laminin, by Thermo Fisher Scientific (3 mentions)
Chir99021, by Merck Group (2 mentions)
Sb431542, by Merck Group (2 mentions)
Dmem f12, by Thermo Fisher Scientific (2 mentions)
Non essential amino acids (neaa), by Merck Group (2 mentions)
Basic fibroblast growth factor (bfgf), by Thermo Fisher Scientific (1 mentions)
Neurobasal media, by Thermo Fisher Scientific (1 mentions)
Vitamin c, by Merck Group (1 mentions)
Advance dmem f12, by Thermo Fisher Scientific (1 mentions)
Ldn193189, by Selleck Chemicals (1 mentions)
Poly ornithine, by Merck Group (1 mentions)
Knockout serum replacement (ksr), by Thermo Fisher Scientific (1 mentions)
Collagenase 4, by Thermo Fisher Scientific (1 mentions)
Ficoll paque plus solution, by GE Healthcare (1 mentions)
Amphotericin b, by PAN Biotech (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Neurobasal medium, by Thermo Fisher Scientific (1 mentions)
Neurobasal, by Thermo Fisher Scientific (1 mentions)
5 protocols using glutmax
1
Cortical Neuroepithelial Stem Cell Differentiation
2
Neural Induction and Maintenance of NESCs
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IVF3.2 and IVF3.3 rESCs and monkey fibroblast-derived iPS line 1.1 were cultured on X-ray-inactivated CF-1 mouse embryonic φμbroblasts (MEFs) in ESCs growth media (DMEM/F12 [1:1] [Invitrogen] containing 15% KSR [Invitrogen] and 5 ng/mL bFGF [Millipore]) (Chen et al., 2015 (link), Li et al., 2005 (link), Sun et al., 2011 (link)).
ESCs or iPS1.1 were digested with Collagenase IV (Gibco), and neural induction was induced by switching from ESC growth media to differentiation media in suspension culture (Advance DMEM/F12 [1:1] [Invitrogen]: Neurobasal media [Invitrogen] [1:1 mixture] supplemented with 1 × N2 [Invitrogen], 1 × B27 [Invitrogen], 10 ng/ml bFGF [Millipore], 3 μM CHIR99021 [Cellagen Technology], 5 μM SB431542 [Cellagen Technology], 0.2 μM compound E, and 0.1 μM LDN193189 [Cellagen Technology]). After 6 days, EBs were transferred to 5 μg/ml laminin (Gibco)-coated plates for attachment culture, and the media were switched to NESCs culture media (Neurobasal media, including B27, N2, and NEAA [Sigma], 1% Glutmax [Sigma], 3 μM CHIR99021, 5 μM SB431542, 10 ng/ml bFGF, and 1,000 U/ml hLIF [Millipore]). To encourage cell propagation, 0.025% trypsin was used to digest NESCs when passaging. NESCs were routinely passaged to 1:8 to 1:16 ratios every 3 to 4 days. For NT formation, NESCs were continually cultured 8 to 10 days before passaging.
ESCs or iPS1.1 were digested with Collagenase IV (Gibco), and neural induction was induced by switching from ESC growth media to differentiation media in suspension culture (Advance DMEM/F12 [1:1] [Invitrogen]: Neurobasal media [Invitrogen] [1:1 mixture] supplemented with 1 × N2 [Invitrogen], 1 × B27 [Invitrogen], 10 ng/ml bFGF [Millipore], 3 μM CHIR99021 [Cellagen Technology], 5 μM SB431542 [Cellagen Technology], 0.2 μM compound E, and 0.1 μM LDN193189 [Cellagen Technology]). After 6 days, EBs were transferred to 5 μg/ml laminin (Gibco)-coated plates for attachment culture, and the media were switched to NESCs culture media (Neurobasal media, including B27, N2, and NEAA [Sigma], 1% Glutmax [Sigma], 3 μM CHIR99021, 5 μM SB431542, 10 ng/ml bFGF, and 1,000 U/ml hLIF [Millipore]). To encourage cell propagation, 0.025% trypsin was used to digest NESCs when passaging. NESCs were routinely passaged to 1:8 to 1:16 ratios every 3 to 4 days. For NT formation, NESCs were continually cultured 8 to 10 days before passaging.
3
Isolation and Culture of Human Granulosa Cells
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Human GCs were isolated and purified as previously
described (22 (link)), which provides the highest percentage
yield of live purified GCs using density gradient
centrifugation. In brief, the isolated follicular fluids
were pooled from differentpatients to reduce the
variability of individual samples. Then, 3000 rpm
centrifugation was performed for 10 minutes to remove
the supernatant and the pellet was resuspended in 2.5
ml of Dulbecco’s Modified Eagle Medium: Nutrient
Mixture F-12 (DMEM/F-12, Gibco, USA). GCs
were isolated using Ficoll-Paque Plus solution (GE
Healthcare, United Kingdom), followed by another
centrifugation at 3000 rpm for 10 min. Next, GCs were
washed and cultured in a complete medium containing
DMEM/F-12 supplemented with 10% heat-inactivated
fetal bovine serum (FBS, Gibco, USA), 100 U/ml of
penicillin (Gibco, USA), 100 mg/ml of (Gibco, USA),
2 mmol/l of glutmax (Sigma-Aldrich, USA), and 2 mg/
ml of amphotericin B (PAN Biotech, Germany) at 37°C
and 5% CO2. The medium was changed after 48 hours.
described (22 (link)), which provides the highest percentage
yield of live purified GCs using density gradient
centrifugation. In brief, the isolated follicular fluids
were pooled from differentpatients to reduce the
variability of individual samples. Then, 3000 rpm
centrifugation was performed for 10 minutes to remove
the supernatant and the pellet was resuspended in 2.5
ml of Dulbecco’s Modified Eagle Medium: Nutrient
Mixture F-12 (DMEM/F-12, Gibco, USA). GCs
were isolated using Ficoll-Paque Plus solution (GE
Healthcare, United Kingdom), followed by another
centrifugation at 3000 rpm for 10 min. Next, GCs were
washed and cultured in a complete medium containing
DMEM/F-12 supplemented with 10% heat-inactivated
fetal bovine serum (FBS, Gibco, USA), 100 U/ml of
penicillin (Gibco, USA), 100 mg/ml of (Gibco, USA),
2 mmol/l of glutmax (Sigma-Aldrich, USA), and 2 mg/
ml of amphotericin B (PAN Biotech, Germany) at 37°C
and 5% CO2. The medium was changed after 48 hours.
4
Neural Progenitor Cell Derivation and Characterization
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NPCs were derived from embryonic stem cell line BG02, provided by Yunnan Key Laboratory of Primate Biomedical Research, Institute of Primate Translational Medicine [41 (link)]. NPCs culture medium contained Neurobasal medium (Gibco, Thermo Fisher Scientific), 1% N2 (Gibco), 2% B27 (Gibco), 1% NEAA (Sigma), 1% Glutmax (Sigma), 10 ng/mL bFGF (1:500; Millipore Ltd., Burlington, MA, USA), and 1000 U/mL LIF (Millipore), 3 μM CHIR99021 (Selleck, Houston, TX, USA), and 5 μM SB431542 (Cellagentec, San Diego, CA, USA). NPCs were cultured with 5 μg/mL laminin (Gibco) in poly-ornithine-coated 12-well plates and were passaged for every three days during the experiment [41 (link)]. Staining was carried out with PAX6 (1:500, Biolegend Covance, Dedham, MA, USA), SOX2 (1:500, Millipore), and Nestin (1:200, Millipore) [42 (link)], which are specific NSC markers. Primary cortical neuron culture was prepared from cerebral cortical tissue from postnatal day one (P1) wild-type C57BL/6 mice. The tissue was digested with 0.25% trypsin for 30 min followed by centrifugation at 1000 rpm for 5 min and filtering cells with 40 μm filter. Astrocytes culture medium contained DMEM (Gibco), 10% fetal bovine serum (FBS, Northvale, NJ, USA), and 1% NEAA (Sigma). Neuron culture medium contained Neurobasal (Gibco), 2% B27 (Gibco), 1% N2 (Gibco), and 1% NEAA (Sigma).
5
HEK293 and HeLa Cell Culture Protocols
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Human embryonic kidney (HEK293) and HeLa cells (Table 1 ) were cultured at 37 °C in a humidified environment containing 5% CO2 in DMEM Glutmax complemented with 10% fetal bovine serum superior (Sigma-Aldrich, St. Louis, MO, USA), penicillin (100 units/mL), and streptomycin (100 units/mL). For chemical treatment experiments, the media of confluent cells was changed to DMEM serum-free for 14–16 h, Forskolin (10 µM) and Isoproterenol (10 µM) were added to the media at the same time as hypoxia treatment, and the PKA inhibitors, Rp-cAMPS (100 µM) and Rp-8-Br-cAMPS (50 µM) were administered to the cells 30 min before lysis. The control and experimental groups’ cells are always generated from the same flask and passage, and they are studied on the same day.
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