Two human RMS cell lines (Rh30; PAX3-FOXO1-positive, RD; PAX3-FOXO1-negative) and HEK293 were purchased from ATCC (#CRL-2061, #CCL-136, and #CRL-1573). Another human RMS cell line, JR1 (PAX3-FOXO1-negative), was kindly provided by Dr. Subramanian (University of Minnesota, MN, USA). Human normal skeletal muscle cell (SkMC) was purchased from Lonza (#CC-2561). All RMS cell lines were maintained in RPMI-1640 (Corning). HEK293 cell line was maintained in DMEM (Corning). Both of the mediums were supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin and 10 mg/ml streptomycin (Corning). SkMC was maintained in Skeletal muscle basal medium (SkBM-2, Lonza) supplemented with hEGF, Dexamethasone, l -glutamine, and gentamicin (Lonza), as well as 10% FBS as described in the manufacturer's instruction. All cells were cultured at 37 °C and 5% CO2. All cell lines were routinely PCR-tested for Mycoplasma. All experiments were performed using cells that have gone through less than 35 passages.
Skbm 2
Manufactured by Lonza
The SkBM-2 is a lab equipment product designed for cell culture applications. It is a compact, tabletop unit that provides a stable and controlled environment for cell growth and maintenance. The SkBM-2 is capable of maintaining precise temperature, humidity, and gas composition settings to support optimal cell culture conditions.
Automatically generated - may contain errors
Lab products found in correlation
Crl 1573, by American Type Culture Collection (1 mentions)
Crl 2061, by American Type Culture Collection (1 mentions)
Penicillin, by Corning (1 mentions)
Streptomycin, by Corning (1 mentions)
Dulbecco modified eagle medium (dmem), by Corning (1 mentions)
Rpmi 1640, by Corning (1 mentions)
L glutamine, by Lonza (1 mentions)
Gentamicin, by Lonza (1 mentions)
Human epidermal growth factor (hegf), by Lonza (1 mentions)
Hek293, by Corning (1 mentions)
Dexamethasone (dex), by Lonza (1 mentions)
Celltiter glo luminescent cell viability assay, by Promega (1 mentions)
Human skeletal muscle myoblasts, by Lonza (1 mentions)
Horse serum, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin amphotericin b, by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
C2c12 cell, by American Type Culture Collection (1 mentions)
Dmem f12, by Thermo Fisher Scientific (1 mentions)
Growth factors, by Lonza (1 mentions)
3 protocols using skbm 2
1
Characterization of Rhabdomyosarcoma Cell Lines
2
Myoblast Proliferation Assay with IGF11-16
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Example 11
Guinea pig myoblasts (Cell Applications) were seeded in a 96-well plate (coated with collagen type I) at 0.1 mL/well (4×103 cells/well) using an SkBM-2 (Lonza, CC-3246) medium containing 1% BSA and were incubated at 37° C. under conditions of 5% CO2. On the next day of the cell seeding, each agent was added to the plate at 25 μL/well and was incubated at 37° C. under conditions of 5% CO2 for 4 days. The amount of intracellular ATP was measured as an index of cell proliferation by CELLTITER-GLO Luminescent Cell Viability Assay (Promega). The supernatant was removed from the 96-well plate subjected to the incubation for 4 days so that the broth in each well was 50 μL, and the plate was then left to stand at room temperature for at least 30 minutes. CELLTITER-GLO reagent was added to the plate at 50 μL/well, followed by reaction for at least 10 minutes. The luminescent signal was then measured with a luminometer (Tristar, Berthold Japan K.K.).
The proliferation activity of guinea pig myoblasts was concentration-dependently enhanced in 0.00005, 0.0005, 0.005, 0.05, 0.5, 5, 50, and 500 nM IGF11-16. The EC50 values of the myoblast proliferative activity of IGF11-16 and IGF-I were 0.004 nM and 0.76 nM, respectively. The results indicate that the activity of IGF11-16 was above 100 times that of IGF-I.
3
Differentiation of Primary Myoblasts
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Primary myoblasts were isolated from hind limb muscles as described previously (37 (link)). Differentiation of primary myoblasts was induced by culturing cells in differentiation medium comprising DMEM (Gibco) supplemented with antibiotics and 5% horse serum (Gibco). C2C12 cells (ATCC) were cultured in DMEM (Gibco) with amphotericin B-penicillin-streptomycin and 10% FBS (Gibco). Human Skeletal Muscle Myoblasts (Lonza Walkersville, Inc.) were cultured in SkBM-2 (Lonza) supplemented with growth factors (Lonza). At 24-48 hr after transfection, differentiation was initiated by changing the medium to differentiation medium containing DMEM/F12 (Gibco), antibiotics, and 2% horse serum (Gibco). To overexpress Caveolin-1 in C2C12 cells, cells were infected with adenovirus expressing Caveolin-1 (Ad-Cav1) or control (Ad-Ctrl) by 30 MOI. After 24 hr of infection, myoblast differentiation was induced by changing the growth medium to differentiation medium.
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