High purity skeletal myocytes express myofilament proteins.

Immunofluorescence staining at day 10 post revival demonstrates robust expression of components of the contractile apparatus such as Desmin (top left), Dystrophin (top right), and Myosin Heavy Chain (bottom left), along with the muscle transcription factor Myogenin (bottom left). Cells also demonstrate expression of Troponin with visible striated fibres and multinucleation (bottom right).

Cells demonstrate timewise gene expression of key myogenic markers.

ioSkeletal Myocytes gene expression. Following reprogramming, ioSkeletal Myocytes downregulate expression of pluripotency markers (A), and begin to express myosin heavy chain isoforms MYH3 and MYH8 (B). Through continued culture, ioSkeletal Myocytes demonstrate expression of mature myosin isoforms MYH7 and MYH1, along with DESMIN, DYSTROPHIN, MYOGENIN, and TITIN (C). Gene expression levels assessed by RT-qPCR (data expressed relative to parental iPSC, normalised to PBGD). Data represents day (Dx) post-thaw.

ioSkeletal Myocytes are available in two vial sizes, tailored to suit your experimental needs with minimal waste.

- Recommended seeding density for ioSkeletal Myocytes is 100,000 cells/cm².
- One Small vial (2.5 x 10⁶ viable cells) can plate a minimum of 0.5 x 24-well plate, 0.75 x 96-well plate, or 1 x 384-well plate.
- One Large vial (5 x 10⁶ viable cells) can plate a minimum of 1 x 24-well plate, 1.5 x 96-well plate, or 2 x 384-well plates.

Cells demonstrate classical myocyte morphology.

Day 1 to 10 post-thawing; 4X magnification; scale bar: 800µm

Immunofluorescence staining of Fast Myosin Skeletal Heavy chain using ab51263 in ioSkeletal Myocytes - Human iPSC-Derived Skeletal Myocytes (ab277612), which were differentiated for 3 (left panel), 5 (middle panel) and 10 days (right panel) post induction.

The cells were fixed with 100% MeOH (5 min) and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated overnight at +4°C with ab51263 at 5 µg/mL and ab6046, rabbit polyclonal to beta Tubulin, at 1/1000 dilution. Cells were then incubated with ab150117, Goat Anti-Mouse IgG H&L (Alexa Fluor^® 488) preadsorbed at 1/1000 dilution (shown in green) and ab150088, Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 594) preadsorbed at 1/1000 dilution (shown in red). Nuclear DNA was labelled with DAPI (shown in blue).

Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a maximum intensity projection of confocal sections is shown. Gamma is adjusted to 1.5 in all channels.

The antibody ab51263 gave comparable results using 4% formaldehyde fixation (10 min).

Immunofluorescence staining of Cardiac Troponin T using ab8295 in ioSkeletal Myocytes - Human iPSC-Derived Skeletal Myocytes (ab277612), which were differentiated for 10 days post induction.

The cells were fixed with 4% formaldehyde (10 min), permeabilized with 0.1% PBS-Tween for 5 mins and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated overnight at +4°C with ab8295 at 5 µg/mL and ab6046, rabbit polyclonal to beta Tubulin, at 1/1000 dilution. Cells were then incubated with ab150117, Goat Anti-Mouse IgG H&L (Alexa Fluor^® 488) preadsorbed at 1/1000 dilution (shown in green) and ab150088, Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 594) preadsorbed at 1/1000 dilution (shown in red). Nuclear DNA was labelled with DAPI (shown in blue).

Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a maximum intensity projection of confocal sections is shown. Gamma is adjusted to 1.5 in all channels.

The antibody ab8295 gave comparable results using MeOH fixation (100%, 5 min).

Immunofluorescence staining of Myogenin using ab124800 in ioSkeletal Myocytes - Human iPSC-Derived Skeletal Myocytes (ab277612), which were differentiated for 3 days post induction.

The cells were fixed with 4% formaldehyde (10 min), permeabilized with 0.1% PBS-Tween for 5 mins and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated overnight at +4°C with ab124800 at 0.5 µg/mL and ab7291, Mouse monoclonal [DM1A] to alpha Tubulin, at 1/1000 dilution. Cells were then incubated with ab150081, Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed at 1/1000 dilution (shown in green) and ab150120, Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) preadsorbed at 1/1000 dilution (shown in red). Nuclear DNA was labelled with DAPI (shown in blue).

Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a maximum intensity projection of confocal sections is shown. Gamma is adjusted to 1.5 in all channels.

The antibody ab124800 gave comparable results using MeOH fixation (100%, 5 min).

Immunofluorescence staining of Myogenin using ab1835 in ioSkeletal Myocytes - Human iPSC-Derived Skeletal Myocytes (ab277612), which were differentiated for 3 days post induction.

The cells were fixed with 4% formaldehyde (10 min), permeabilized with 0.1% PBS-Tween for 5 mins and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated overnight at +4°C with ab1835 at 1 µg/mL and ab6046, rabbit polyclonal to beta Tubulin, at 1/1000 dilution. Cells were then incubated with ab150117, Goat Anti-Mouse IgG H&L (Alexa Fluor^® 488) preadsorbed at 1/1000 dilution (shown in green) and ab150088, Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 594) preadsorbed at 1/1000 dilution (shown in red). Nuclear DNA was labelled with DAPI (shown in blue).

Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a maximum intensity projection of confocal sections is shown. Gamma is adjusted to 1.5 in all channels.

The antibody ab1835 gave comparable results using MeOH fixation (100%, 5 min).

Immunofluorescence staining of Actinin/ACTN1 using ab68194 in ioSkeletal Myocytes - Human iPSC-Derived Skeletal Myocytes (ab277612), which were differentiated for 3 (left panel), 5 (middle panel) and 10 days (right panel) post induction.

The cells were fixed with 100% MeOH (5 min) and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated overnight at +4°C with ab68194 at 0.5 µg/mL and ab7291, Mouse monoclonal [DM1A] to alpha Tubulin, at 1/1000 dilution. Cells were then incubated with ab150081, Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed at 1/1000 dilution (shown in green) and ab150120, Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) preadsorbed at 1/1000 dilution (shown in red). Nuclear DNA was labelled with DAPI (shown in blue).

Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a maximum intensity projection of confocal sections is shown. Gamma is adjusted to 1.5 in all channels.

Immunofluorescence staining of Actinin/ACTN1 using ab68194 in ioSkeletal Myocytes - Human iPSC-Derived Skeletal Myocytes (ab277612), which were differentiated for 3 (left panel), 5 (middle panel) and 10 days (right panel) post induction.

The cells were fixed with 4% formaldehyde (10 min), permeabilized with 0.1% PBS-Tween for 5 mins and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated overnight at +4°C with ab68194 at 0.5 µg/mL and ab7291, Mouse monoclonal [DM1A] to alpha Tubulin, at 1/1000 dilution. Cells were then incubated with ab150081, Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed at 1/1000 dilution (shown in green) and ab150120, Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) preadsorbed at 1/1000 dilution (shown in red). Nuclear DNA was labelled with DAPI (shown in blue).

Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a maximum intensity projection of confocal sections is shown. Gamma is adjusted to 1.5 in all channels.

Immunofluorescence staining of Actinin/ACTN1 using ab18061 in ioSkeletal Myocytes - Human iPSC-Derived Skeletal Myocytes (ab277612), which were differentiated for 10 days post induction.

The cells were fixed with 100% MeOH (5 min) and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated overnight at +4°C with ab18061 at 5 µg/mL and ab6046, rabbit polyclonal to beta Tubulin, at 1/1000 dilution. Cells were then incubated with ab150117, Goat Anti-Mouse IgG H&L (Alexa Fluor^® 488) preadsorbed at 1/1000 dilution (shown in green) and ab150088, Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 594) preadsorbed at 1/1000 dilution (shown in red). Nuclear DNA was labelled with DAPI (shown in blue).

Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a maximum intensity projection of confocal sections is shown. Gamma is adjusted to 1.5 in all channels.

Immunofluorescence staining of Cardiac Troponin T using ab10214 in ioSkeletal Myocytes - Human iPSC-Derived Skeletal Myocytes (ab277612), which were differentiated for 10 days post induction.

The cells were fixed with 100% MeOH (5 min) and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated overnight at +4°C with ab10214 at 1 µg/mL and ab6046, rabbit polyclonal to beta Tubulin, at 1/1000 dilution. Cells were then incubated with ab150117, Goat Anti-Mouse IgG H&L (Alexa Fluor^® 488) preadsorbed at 1/1000 dilution (shown in green) and ab150088, Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 594) preadsorbed at 1/1000 dilution (shown in red). Nuclear DNA was labelled with DAPI (shown in blue).

Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a maximum intensity projection of confocal sections is shown. Gamma is adjusted to 1.5 in all channels.

The antibody ab10214 also gave a positive staining using 4% formaldehyde fixation (10 min).

Immunofluorescence staining of Cardiac Troponin T using ab209813 in ioSkeletal Myocytes - Human iPSC-Derived Skeletal Myocytes (ab277612), which were differentiated for 10 days post induction.

The cells were fixed with 4% formaldehyde (10 min), permeabilized with 0.1% PBS-Tween for 5 mins and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated overnight at +4°C with ab209813 at 0.02 µg/mL and ab7291, Mouse monoclonal [DM1A] to alpha Tubulin, at 1/1000 dilution. Cells were then incubated with ab150081, Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed at 1/1000 dilution (shown in green) and ab150120, Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) preadsorbed at 1/1000 dilution (shown in red). Nuclear DNA was labelled with DAPI (shown in blue).

Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a maximum intensity projection of confocal sections is shown. Gamma is adjusted to 1.5 in all channels.

The antibody ab209813 also gave a positive staining using MeOH fixation (100%, 5 min).

Immunofluorescence staining of Desmin using ab32362 in ioSkeletal Myocytes - Human iPSC-Derived Skeletal Myocytes (ab277612), which were differentiated for 10 days post induction.

The cells were fixed with 100% MeOH (5 min) and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated overnight at +4°C with ab32362 at 0.02 µg/mL and ab7291, Mouse monoclonal [DM1A] to alpha Tubulin, at 1/1000 dilution. Cells were then incubated with ab150081, Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed at 1/1000 dilution (shown in green) and ab150120, Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) preadsorbed at 1/1000 dilution (shown in red). Nuclear DNA was labelled with DAPI (shown in blue).

Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a maximum intensity projection of confocal sections is shown. Gamma is adjusted to 1.5 in all channels.

The antibody ab32362 gave comparable results using 4% formaldehyde fixation (10 min).

Immunofluorescence staining of Desmin using ab15200 in ioSkeletal Myocytes - Human iPSC-Derived Skeletal Myocytes (ab277612), which were differentiated for 10 days post induction.

The cells were fixed with 4% formaldehyde (10 min), permeabilized with 0.1% PBS-Tween for 5 mins and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated overnight at +4°C with ab15200 at 0.1 µg/mL and ab7291, Mouse monoclonal [DM1A] to alpha Tubulin, at 1/1000 dilution. Cells were then incubated with ab150081, Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed at 1/1000 dilution (shown in green) and ab150120, Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) preadsorbed at 1/1000 dilution (shown in red). Nuclear DNA was labelled with DAPI (shown in blue).

Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a maximum intensity projection of confocal sections is shown. Gamma is adjusted to 1.5 in all channels.

The antibody ab15200 gave comparable results using MeOH fixation (100%, 5 min).

Critically for metabolic studies, data demonstrates expression of the insulin regulated glucose transporter GLUT4 (Part 2)

Immunocytochemistry at Day 7 post-revival demonstrates expression of GLUT4 in peri-nuclear regions, and striations, in the ioSkeletal Myocytes.

Image courtesy of Dougall Norris & Daniel Fazakerley, Wellcome-MRC Institute of Metabolic Science.

Critically for metabolic studies, data demonstrates expression of the insulin regulated glucose transporter GLUT4 (Part 1)

RT-qPCR at Day 10 post-revival demonstrates expression of GLUT4 in the ioSkeletal Myocytes, compared to undifferentiated hiPSCs and ioGlutamatergic Neurons.

Immunofluorescence staining of GLUT4 using ab33780 in ioSkeletal Myocytes - Human iPSC-Derived Skeletal Myocytes (ab277612), which were differentiated for 3 (left panel), 5 (middle panel) and 10 days (right panel) post induction.

The cells were fixed with 100% MeOH (5 min) and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated overnight at +4°C with ab33780 at 5 µg/mL and ab7291, Mouse monoclonal [DM1A] to alpha Tubulin, at 1/1000 dilution. Cells were then incubated with ab150081, Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed at 1/1000 dilution (shown in green) and ab150120, Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) preadsorbed at 1/1000 dilution (shown in red). Nuclear DNA was labelled with DAPI (shown in blue).

Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a maximum intensity projection of confocal sections is shown. Gamma is adjusted to 1.5 in all channels.

Critically for metabolic studies, data demonstrates expression of the insulin regulated glucose transporter GLUT4 (Part 3)

Western blotting of differentiated 3T3-L1 adipocytes and maturing ioSkeletal Myocytes demonstrates GLUT4 expression in a time-dependent manner.

Image courtesy of Dougall Norris & Daniel Fazakerley, Wellcome-MRC Institute of Metabolic Science.

ioSkeletal Myocytes cells arrive ready to plate. A simple one-medium protocol generates fully differentiated and mature skeletal myocytes.

The 3 phase protocol for generating ioSkeletal Myocytes:
1. Induction (carried out at bit.bio)
2. Stabilization for 3 days with Doxycycline
3. Maintenance during which the skeletal myocytes mature.