HEMA 3 Staining: A Simple Alternative for the Assessment of Myoblast Differentiation

Abstract

Skeletal muscle tissue regeneration requires quiescent satellite cell activation, proliferation, and differentiation. Regenerative capacity of satellite cells can be studied in vitro by differentiating under low‐serum conditions (2% to 5%) to form multinucleated myotubes. Myotubes are fixed and stained, and indices of differentiation are quantified. Jenner and Giemsa stains are typically used for myotube staining; however, this staining process can be variable depending on factors such as stain pH, staining time, and time since stain preparation. This article includes protocols for myoblast isolation, proliferation, and differentiation in vitro; Jenner‐Giemsa staining; HEMA 3 staining; and quantification. Representative images using each staining method and quantification are included. The protocols identify critical steps and considerations for cell culture and each staining method and provide an even simpler alternative to Jenner‐Giemsa staining. © 2019 by John Wiley & Sons, Inc. Basic Protocol 1: Primary myoblast isolation Alternate Protocol 1: Plating cryopreserved myoblasts Basic Protocol 2: Myoblast passage and expansion Basic Protocol 3: Myoblast differentiation Basic Protocol 4: HEMA 3 staining Alternate Protocol 2: Jenner‐Giemsa staining Basic Protocol 5: Quantification of myotube density Basic Protocol 6: Quantification of fusion index Basic Protocol 7: Quantification of myotubes per field

Spexin Promotes the Proliferation and Differentiation of C2C12 Cells In Vitro—The Effect of Exercise on SPX and SPX Receptor Expression in Skeletal Muscle In Vivo

“…After the 6th day of differentiation, the cells were fixed by methanol. The staining protocol was then performed as previously described [ 21 ]. In brief, the fusion index was calculated by staining the fixed cell and determining the number of nuclei in a myotubule compared to the total number of nuclei in the sample using an inverted Delta Optical IB-100 light microscope.…”

Ethanol‐Impaired Myogenic Differentiation is Associated With Decreased Myoblast Glycolytic Function

“…Primary myoblasts were isolated from vastus lateralis muscle samples as previously described (Levitt et al, 2019; Simon et al, 2014, 2017). Samples were washed 3 times in Ham’s F‐12 nutrient mixture (GE Healthcare Life Sciences, Marlborough, MA), minced, and trypsin‐digested (0.25% trypsin EDTA diluted 1:4 in Ham’s F‐12).…”

“…Myoblast cell culture was performed according to our previously published detailed methods (Adler et al, 2019; Levitt et al, 2019). Myoblasts were seeded in growth media at a density of 2.8 × 10 3 cells per cm 2 on collagen‐coated 6‐well plates containing either 0 or 50 mM EtOH and maintained in cell culture incubators under standard culture conditions (5% CO 2 , 37°C) until reaching 70 to 80% confluence (3 days).…”

“…Fixed cells were stored covered at room temperature until staining. Fixed cells were stained using our previously published HEMA 3 method (Levitt et al, 2019). Cells were treated with HEMA 3 solution I (Fisher HealthCare, Inc., Waltham, MA) for 10 minutes at room temperature.…”

“…For each image, total nuclei and nuclei fused into myotubes were counted using ImageJ. Myotubes per field were calculated as the number of cells with 3 or more nuclei and fusion index was calculated as the percentage of all nuclei fused into myotubes (Adler et al, 2019; Levitt et al, 2019; Simon et al, 2014). Final calculations of fusion index, myotubes per field, and total nuclei across the 15 images per well were averaged for analysis.…”