Memα basal medium

Manufactured by Thermo Fisher Scientific

MEMα basal medium is a cell culture medium designed to support the growth and maintenance of a variety of cell types. It provides essential nutrients, vitamins, and salts required for cell metabolism and proliferation.

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4 protocols using memα basal medium

Isolation and Culture of Mouse Spermatogonial Stem Cells

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We isolated and purified SSCs from neonatal mouse testes (6 days old, C57BL/6) following a previously described protocol [48 (link)]. We cultured SSCs on STO feeder cells in MEMα basal medium (Invitrogen) supplemented with 10% FBS (Life Technologies), 1 mM sodium pyruvate (Amresco), 2 mM L-glutamine (Amresco), 50 μM β-mercaptoethanol (Biotech), 1 mM NEAA (Invitrogen), 20 ng/ml mouse EGF (PeproTech), 10 ng/ml human bFGF (PeproTech), 10 ng/ml mouse GDNF (PeproTech), 10 ng/ml ESGRO (Santa Cruz Biotechnology), and 100 μg/ml transferrin (Sigma). Spermatogonial stem cells were identified by immunofluorescence staining and RT-PCR (Additional file 1: Figure S2).

Hu X., Wang H., Tian G.G., Hou C., Xu B., Zhao X., Zhao Y., Fang Q., Li X., He L., Chen X., Li S, & Wu J. (2022). Offspring production of haploid spermatid-like cells derived from mouse female germline stem cells with chromatin condensation. Cell & Bioscience, 12, 5.

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Isolation and Purification of Fetal Germ Stem Cells

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We isolated and purified FGSCs from neonatal mouse ovaries (6 days old, C57BL/6) following a previously described protocol [31 (link)]. To trace cells during in vivo and in vitro transdifferentiation, FGSCs were derived from actin-GFP mice. For immunofluorescence and infection with lentivirus carrying STRA8-EGFP or PRM1-EBFP plasmids, FGSCs were derived from wild-type mice. We cultured FGSCs on SIM-6-thiogunanie-oualiain(STO) feeder cells in MEMα basal medium (Invitrogen) supplemented with 10% fetal bovine serum (FBS) (Life Technologies), 1 mM sodium pyruvate (Amresco), 2 mML-glutamine (Amresco), 50 μM β-mercaptoethanol (Biotech), 1 mMnonessential amino acids (NEAA) (Invitrogen), 20 ng/ml mouse epidermal growth factor (EGF) (PeproTech), 10 ng/ml human basic fibroblast growth factor (bFGF) (PeproTech), 10 ng/ml mouse glial cell line-derived neurotrophic factor (GDNF) (PeproTech), and 10 ng/ml ESGRO (mouse leukemia inhibitory factor, LIF) (Santa Cruz Biotechnology). FGSCs were identified by immunofluorescence staining (Positive expression of FRAGILIS, MVH, and OCT4; negative expression of PLZF.) and RT-PCR (Additional file 1: Figure S1).

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Differentiation of hESCs to hMSCs

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SIRT3^+/+ hESCs (H9 hESCs, WiCell Research) and SIRT3^–^/^- hESCs were cultured on mouse embryonic fibroblast (MEF, inactivated with mitomycin C) feeder cells in hESC culture medium (DMEM/F12 (Thermo Fisher Scientific), supplemented with 20% Knockout Serum Replacement (Thermo Fisher Scientific), 1% penicillin/streptomycin (Thermo Fisher Scientific), 0.1 mM non-essential amino acids (NEAA, Thermo Fisher Scientific), 2 mM GlutaMAX (Thermo Fisher Scientific), 55 μM β-mercaptoethanol (Thermo Fisher Scientific), and 10 ng/ml bFGF (Joint Protein Central)), or maintained on Matrigel-coated plates with mTeSR medium. hMSCs differentiated from hESCs and primary hMSCs were cultured in hMSC culture medium (Minimum Essential Medium α (MEMα) basal medium (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS, Gemcell, Cat# 100-500, Lot# A77E01F), 0.1 mM NEAA, 1% penicillin/streptomycin, and 1 ng/ml bFGF).

Diao Z., Ji Q., Wu Z., Zhang W., Cai Y., Wang Z., Hu J., Liu Z., Wang Q., Bi S., Huang D., Ji Z., Liu G.H., Wang S., Song M, & Qu J. (2021). SIRT3 consolidates heterochromatin and counteracts senescence. Nucleic Acids Research, 49(8), 4203-4219.

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hESC and hMSC Culture Protocols

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ZKSCAN3^+/+ and ZKSCAN3^-/- hESCs (derived from Line H9 from WiCell Research Institute) were cultured either on mitomycin C-inactivated mouse embryonic fibroblast (MEF) feeders in hESC medium including DMEM/F12 (Thermo Fisher Scientific), 20% Knockout Serum Replacement (Thermo Fisher Scientific), 1% penicillin/streptomycin (Thermo Fisher Scientific), 2 mM GlutaMAX (Thermo Fisher Scientific), 0.1 mM non-essential amino acids (NEAAs, Thermo Fisher Scientific), 55 μM β-mercaptoethanol (Thermo Fisher Scientific), and 10 ng/ml bFGF (Joint Protein Central, Incheon, Korea) or on Matrigel (BD Biosciences)-coated plates in mTeSR medium (STEMCELL Technologies). hMSCs (hESC-derived or primary hMSCs) were cultured in hMSC medium which contained MEMα basal medium (Thermo Fisher Scientific), 10% fetal bovine serum (FBS, Gemcell), 1% penicillin/streptomycin (Thermo Fisher Scientific), 0.1 mM NEAAs (Thermo Fisher Scientific), and 1 ng/ml bFGF (Joint Protein Central, Incheon, Korea). HEK293T cells were cultured in high glucose DMEM (Hyclone) supplemented with 10% FBS (Gibco). There was no mycoplasma contamination observed during cell culture.

Hu H., Ji Q., Song M., Ren J., Liu Z., Wang Z., Liu X., Yan K., Hu J., Jing Y., Wang S., Zhang W., Liu G.H, & Qu J. (2020). ZKSCAN3 counteracts cellular senescence by stabilizing heterochromatin. Nucleic Acids Research, 48(11), 6001-6018.

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