PHSats were isolated with hypothermic treatment as described previously.27 (link),28 (link) Cells were cultured in Skeletal Muscle Growth Medium (SMCGM) (Provitro, Berlin, Germany) enriched with supplement mix (Provitro) at 37°C in a humidified incubator with 5% CO2. For passaging, cells were washed with Dulbecco’s phosphate-buffered saline (DPBS) (Thermo Fisher Scientific, Waltham, MT) and detached with TrpLE Express (Thermo Fisher) at 37°C for 5 min.
Supplement mix
Manufactured by Provitro
Sourced in Germany
The Supplement mix is a lab equipment product that provides a precise combination of essential nutrients and compounds. Its core function is to facilitate the preparation of standardized nutritional supplements for research and development purposes.
Automatically generated - may contain errors
Lab products found in correlation
Smcgm, by Provitro (2 mentions)
Skeletal muscle growth medium, by Provitro (2 mentions)
Trple express, by Thermo Fisher Scientific (1 mentions)
Human umbilical vein endothelial cells (huvec), by Provitro (1 mentions)
Trypsin edta, by Provitro (1 mentions)
Dulbecco s phosphate buffered saline dpbs, by Thermo Fisher Scientific (1 mentions)
Tryple express, by Thermo Fisher Scientific (1 mentions)
Dulbecco s modified eagle medium dmem, by Harvard Bioscience (1 mentions)
Fetal calf serum (fcs), by Harvard Bioscience (1 mentions)
Polyethylenimine (pei), by Merck Group (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Lipofectamine rnaimax, by Thermo Fisher Scientific (1 mentions)
Endothelial cell growth supplement (ecgs), by Corning (1 mentions)
Penicillin, by Provitro (1 mentions)
Streptomycin, by Provitro (1 mentions)
Amphotericin, by Provitro (1 mentions)
Streptomycin, by Harvard Bioscience (1 mentions)
Dulbecco modified eagle medium (dmem), by Harvard Bioscience (1 mentions)
Penicillin, by GE Healthcare (1 mentions)
Streptomycin, by GE Healthcare (1 mentions)
5 protocols using supplement mix
1
Isolation and Culture of PHSats
2
In Vitro Endothelial Cell Experiment
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In vitro experiments were performed using commercial human umbilical vein endothelial cells (HUVEC) Cat.-No.: 121 0113; Provitro AG, Berlin, Germany), cultured in endothelial cell proliferation medium, basal (ECPM) according to the manufacturer’s instructions (Cat.-Nr.: 201 0001; Provitro AG) and supplements [Supplement-Mix (Cat.-Nr.: 218 0001) with antibiotics (Cat.-Nr.: 236 0350), Provitro AG]. No special permission regarding ethics approval for this study is required to use cells of human origin.
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In vitro experiments were performed using commercial human umbilical vein endothelial cells (HUVEC) Cat.-No.: 121 0113; Provitro AG, Berlin, Germany), cultured in endothelial cell proliferation medium, basal (ECPM) according to the manufacturer’s instructions (Cat.-Nr.: 201 0001; Provitro AG) and supplements [Supplement-Mix (Cat.-Nr.: 218 0001) with antibiotics (Cat.-Nr.: 236 0350), Provitro AG]. No special permission regarding ethics approval for this study is required to use cells of human origin.
3
Isolation and Culture of Primary Murine Satellite Cells
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MuSC isolation was performed as described.13 (link),20 ,34 (link) Primary MuSC populations used in this study are listed in Tables 1 and S1 . Primary MuSCs were cultured in Skeletal Muscle Cell Growth Medium (SMCGM, Provitro, Germany) enriched with supplement mix (Provitro) at 37°C in at humidified incubator with 5% CO2. For cell passaging, MuSCs were washed with Dulbecco’s phosphate-buffered saline (DPBS) (Thermo Fisher Scientific) and detached with 0.25% trypsin-EDTA (Provitro) or TrypLE Express (Gibco, Thermo Fisher Scientific) at 37°C for 5 min.
4
Cultivation and Transfection of Myoblast and HEK293T Cells
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C2C12 mouse myoblasts and HEK293T cells were cultivated in Dulbecco’s modified Eagle Medium (DMEM; Biochrom AG) supplemented with 10% fetal calf serum (FCS; Biochrom AG), 2 mM L-glutamine and penicillin (100 units/ml)/streptomycin (10 µg/ml) (PAA) at 37°C and 10% or 5% CO2, respectively. Immortalised human myoblasts were cultured in skeletal muscle growth medium (Provitro) supplemented with supplement mix (Provitro), 50 ng/ml amphotericin, 50 µg/ml gentamicin, 10% FCS, 2 mM L-glutamine and penicillin (100 units/ml)/streptomycin (10 µg/ml) at 37°C and 5% CO2.
For transient transfection of HEK293T cells polyethylenimine (PEI; Sigma-Aldrich) was used. C2C12 cells were transfected using Lipofectamine2000 (Invitrogen) according to manufacturer’s instructions. For siRNA-mediated knockdown of human IRS4 Lipofectamine RNAiMAX (Invitrogen) was used according to manufacturer’s instructions; as control non-targeting siRNA was used (for detailed information see Supplementary Methods). In short, cells were transfected with 25 nM siRNA on two consecutive days and experiments were performed 72 h post-transfection.
For transient transfection of HEK293T cells polyethylenimine (PEI; Sigma-Aldrich) was used. C2C12 cells were transfected using Lipofectamine2000 (Invitrogen) according to manufacturer’s instructions. For siRNA-mediated knockdown of human IRS4 Lipofectamine RNAiMAX (Invitrogen) was used according to manufacturer’s instructions; as control non-targeting siRNA was used (for detailed information see Supplementary Methods). In short, cells were transfected with 25 nM siRNA on two consecutive days and experiments were performed 72 h post-transfection.
5
Culturing Diverse Cell Lines for Experiments
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HEK293T, MCF7, and C2C12 cells were grown in DMEM (Biochrom AG) supplemented with 10% fetal calf serum (FCS; Biochrom AG), 2 mM l -glutamine, and penicillin (100 U/ml)/streptomycin (10 µg/ml) (PAA Laboratories) at 37°C and 10% (C2C12) or 5% CO2. Immortalized human myoblasts were cultured in skeletal muscle growth medium (Provitro) supplemented with supplement mix (Provitro), 50 ng/ml amphotericin, 50 µg/ml gentamicin, 10% FCS, 2 mM l -glutamine, and penicillin (100 U/ml)/streptomycin (10 µg/ml) at 37°C and 5% CO2. hFOBs (1.19) were cultured in a 1:1 mixture of DMEM and Ham’s F12 medium supplemented with 10% FCS, 2 mM l -glutamine, penicillin (100 U/ml)/streptomycin (10 µg/ml), and 0.3 mg/ml G418 (Biochrom AG) at 34°C with 5% CO2 to keep them in a proliferative state. HUVECs were a kind gift from M. Lorenz and V. Stangl (Charité Universitätsmedizin, Berlin, Germany) and cultured on gelatin-coated tissue culture ware in M199 medium supplemented with 20% FCS, 50 µg/ml endothelial cell growth supplement (Corning), 25 µg/ml heparin, 2 mM l -glutamine, and penicillin (100 U/ml)/streptomycin (10 µg/ml) at 37°C and 5% CO2. HUVECs were used at passage 3 in all experiments. Unless stated otherwise, all cells were starved for 5 h prior to stimulation with their respective growth medium, without FCS supplement, containing 2 mM l -glutamine and penicillin/streptomycin.
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