De-identified muscle samples were collected from patients during surgical treatment at Stanford University Hospital in accordance with the Human Subjects Committee of Stanford University guidelines. Primary human myoblasts were isolated from these samples as previously described30 (link). Myoblasts were plated onto 15-cm polystyrene dishes coated overnight at 37°C with a 22 μg/mL collagen solution (Sigma, C-8919). The cells were cultured in skeletal muscle cell growth medium (SkGM) supplemented with human epidermal growth factor (EGF), dexamethasone, gentamicin/amphotericin-B, fetuin, insulin, and bovine serum albumin provided in the SkGM Bullet Kit (Lonza, CC-3160); cell medium was changed every other day. HyQtase (Thermo Scientific, SV3003001) was used to dissociate cells from polystyrene dishes. All migration studies were performed in a modified version of SkGM where EGF was removed. Myoblasts were not used for experiments beyond passage six.
Skgm bullet kit
Manufactured by Lonza
Sourced in Switzerland
The SkGM Bullet Kit is a laboratory equipment product designed for stem cell research. It provides a standardized system for the isolation, culture, and expansion of stem cells. The kit includes essential components and reagents to support basic stem cell growth and maintenance activities in a controlled laboratory setting.
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3 protocols using skgm bullet kit
1
Isolation and Culture of Primary Human Myoblasts
2
Primary Human Skeletal Muscle Cells
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Primary human skeletal muscle myoblasts (HSMM; from post gestational tissue, usually from the quadriceps or psoas tissue) and primary human skeletal muscle cells (SkMC; isolated from the upper arm or leg skeletal muscle tissue) were propagated as specified by the supplier (Lonza, Walkersville, MD) and maintained in Clonetics SkGM-2 and SkGM Bullet kit (Lonza), respectively. HEK293T cells were from the ATCC (Manassas, VA) and maintained in DMEM + 10% FCS.
3
Isolation and Differentiation of Primary Human Skeletal Muscle Cells
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Primary human skeletal muscle cells were isolated, cultured, and differentiated by established methods [16 (link), 17 (link)] as described in [18 (link)]. In brief, satellite cells from human rectus abdominis muscle biopsies were released from myotubes by enzymatic digestion. Contaminating fibroblasts were removed by adhesion of myoblasts to CD56 (Leu 19; Becton Dickinson, Franklin Lakes, MJ, USA), which was immobilized onto magnetic beads (CELLection Pan Mouse IgG Kit, Life Technologies). The purified myoblast population was plated on collagen-coated dishes and expanded in skeletal muscle cell growth medium (SkGM Bullet Kit, Lonza, Basel, Switzerland). SH-SY5Y cells were obtained from ATCC and cultured in DMEM/F12 1 : 1 (Invitrogen). C2C12 cells were cultured in DMEM. Culture media were supplemented with 10% fetal bovine serum and 0.01% penicillin/streptomycin.
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