Knockout serum replacement formulation

Manufactured by Thermo Fisher Scientific

KnockOut serum replacement formulation is a cell culture medium supplement designed to support the growth and maintenance of various cell types without the need for animal-derived serum. It provides essential nutrients, growth factors, and other components to promote cell proliferation and differentiation.

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Knockout serum (54 citations) Serum replacement (41 citations) KnockOutTM Serum Replacement (28 citations)

3 protocols using knockout serum replacement formulation

Expansion and Differentiation of Human Primary Myoblasts

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Human primary myoblast cell lines were obtained from the University of Rochester biorepository (http://www.urmc.rochester.edu/fields-center/) and were expanded and maintained in DMEM/F-10 (#31550 Gibco/Life Technologies, Bleiswijk, The Netherlands) supplemented with 20% heat inactivated fetal bovine serum (FCS #10270 Gibco), 1% penicillin/streptomycin (#15140 Gibco), 10 ng/mL rhFGF (#G5071 Promega, Leiden, The Netherlands) and 1 μM dexamethasone (#D2915 Sigma-Aldrich, Zwijndrecht, The Netherlands). Differentiation into myotubes was started at 80% confluency by serum starvation in DMEM/F-12 Glutamax (#31331, Gibco) supplemented with 2% KnockOut serum replacement formulation (#10828 Gibco) for 36 h. All samples and their characteristics used for our study are listed in Additional file
1: Table S1. Rhabdomyosarcoma TE-671 were maintained in DMEM (#31966) supplemented with 10% FCS and 1% P/S (all Gibco).

Thijssen P.E., Balog J., Yao Z., Pham T.P., Tawil R., Tapscott S.J, & Van der Maarel S.M. (2014). DUX4 promotes transcription of FRG2 by directly activating its promoter in facioscapulohumeral muscular dystrophy. Skeletal Muscle, 4, 19.

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Standardized primary myoblast culture

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Human primary myoblast cell lines were originating from the University of Rochester bio repository (http://www.urmc.rochester.edu/fields-center/). Muscle samples were obtained after subjects were consented under a protocol approved by the institutional review board at the University of Rochester. Myoblasts were cultured in DMEM/F-10 media (#31550, Gibco/Life Technologies, Bleiswijk, The Netherlands) supplemented with 20% heat inactivated fetal bovine serum (FBS #10270, Gibco/Life Technologies), 1% penicillin/streptomycin (#15140, Gibco/Life Technologies) and 10ng/ml rhFGF (#G5071, Promega, Leiden, The Netherlands) and 1μM dexamethasone (#D2915, Sigma-Aldrich, Zwijndrecht, The Netherlands) was added to the medium. Myoblasts were fused at 80% confluency by culturing them in DMEM/F-12 Glutamax media (#31331, Gibco/Life Technologies) containing 1% penicillin and streptomycin and 2% KnockOut serum replacement formulation (#10828, Gibco/Life Technologies) for 36 h. Human control fibroblast cell lines were maintained in DMEM/F-12, supplemented with 20% FBS, 1% penicillin and streptomycin, 10 mM HEPES (#15630#, Gibco/Life Technologies) and 1mM sodium pyruvate (##11360, Gibco/Life Technologies). Used cell lines, D4Z4 allele information, and experimental use are listed in Table S1.

Balog J., Thijssen P.E., Shadle S., Straasheijm K.R., van der Vliet P.J., Krom Y.D., van den Boogaard M.L., de Jong A., F Lemmers R.J., Tawil R., Tapscott S.J, & van der Maarel S.M. (2015). Increased DUX4 expression during muscle differentiation correlates with decreased SMCHD1 protein levels at D4Z4. Epigenetics, 10(12), 1133-1142.

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Optimizing Myoblast Culture and Differentiation

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Primary and immortalized myoblasts were cultured in DMEM/F-10 medium (#31550, Gibco/Life Technologies) supplemented with 20% fetal bovine serum (FBS #10270, Gibco/Life Technologies), 1% penicillin/streptomycin (Pen/Strep #15140, Gibco/Life Technologies), with addition of 10 ng/ml rhFGF (#G5071, Promega) and 1 μM dexamethasone (#D2915, Sigma-Aldrich). Myoblasts were fused at 80% confluency by replacing growth medium with DMEM/F-12 Glutamax medium (#31331, Gibco/Life Technologies) containing 1% penicillin/streptomycin and 2% KnockOut serum replacement formulation (#10828, Gibco/Life Technologies) for 2–5 days depending on the cell line. The HEK293T cells were grown in Gibco DMEM, High Glucose, Pyruvate (#119950, Gibco/Life Technologies) with addition of 10% FBS and 1% penicillin/streptomycin. Primary fibroblasts were cultured in DMEM/F-12 GlutaMAX™ Supplement (Gibco, #10565018) supplemented with 20% FBS, 1% penicillin/streptomycin, 10 mM HEPES (Gibco, #15630056) and 1 mM Sodium Pyruvate (Gibco, #11360070). The human colon carcinoma HCT116 (WT and DKO) cell lines were grown in McCoy’s 5 A medium (Thermo Fisher Scientific, #16600082) supplemented with 10% FBS and 1% penicillin/streptomycin. Additional information about cell lines is provided in Supplementary Table 1.

Šikrová D., Testa A.M., Willemsen I., van den Heuvel A., Tapscott S.J., Daxinger L., Balog J, & van der Maarel S.M. (2023). SMCHD1 and LRIF1 converge at the FSHD-associated D4Z4 repeat and LRIF1 promoter yet display different modes of action. Communications Biology, 6, 677.

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