c-pace ep cell culture stimulator with carbon electrodes  (IonOptix)

Journal: Frontiers in Physiology

Article Title: High-resolution analyses of the secretomes from murine C2C12 cells and primary human skeletal muscle cells reveal distinct differences in contraction-regulated myokine secretion

doi: 10.3389/fphys.2025.1549316

Figure Lengend Snippet: Bottom-up proteomics workflow of secretome and proteome analyses. Murine C2C12 and primary human myotubes were subjected to acute low-frequency EPS. CM from stimulated and unstimulated cells were collected for secretome analysis, whereas only unstimulated cells were lyzed and harvested for cellular proteome analysis. Proteins were digested using trypsin/Lys-C mix prior to high-resolution LC-MS analysis. Samples from C2C12 cells ( n = 3) are demonstrated in blue, while samples from HSkMCs from three different donors ( n = 3) are shown in green. CON: control, CM: conditioned media, EPS: electrical pulse stimulation, LC: liquid chromatography, MS: mass spectrometry.

Article Snippet: Myotubes were stimulated in serum-free medium with EPS (C-Pace EP, IonOptix LLC) for 6 hours with 2 m pulses at 40 V (HSkMCs) and 11.5 V (C2C12) following the most frequently used established protocols for these cell types ( ).

Techniques: Liquid Chromatography with Mass Spectroscopy, Control, Liquid Chromatography, Mass Spectrometry

Journal: Frontiers in Physiology

Article Title: High-resolution analyses of the secretomes from murine C2C12 cells and primary human skeletal muscle cells reveal distinct differences in contraction-regulated myokine secretion

doi: 10.3389/fphys.2025.1549316

Figure Lengend Snippet: EPS-induced differentially regulated proteins in murine und human muscle secretome. Cell lysates from C2C12 myotubes (A) or from human myotubes from three individuals (B) were analyzed for changes in phosphorylation of AMPKα following EPS by immunoblotting with AMPKα-Thr 172 specific antibody. IL-6 protein secreted in the CM of C2C12 (C) and human myotubes (D) was measured using multiplex immunoassay. Volcano plot analysis of 172 downregulated and 346 upregulated myokines in the C2C12 secretome (E) Volcano plot analysis of 199 downregulated and 137 upregulated myokines in the HSkMCs secretome (F) . Bioinformatic prediction analysis of differentially significantly regulated proteins from C2C12 secretome (G) or human secretome (H) . Gene Ontology cellular component (GOCC) analysis of differentially significantly regulated proteins classified as SP+, SP−, Outcyte+ from C2C12 secretome (I) or human secretome (J) . Phosphorylation signals in immunoblot analysis were normalized to GAPDH protein abundance and total AMPKα. Data are means ± SEM from C2C12 cells ( n = 6) (A) and HSkMC (B) from three individuals ( n = 3) and were analyzed by unpaired t-test (Welch´s correction), * p < 0.05 vs. CON. Data from C2C12 cells ( n = 3) are shown in blue and data of HSkMCs from three subjects ( n = 3) are presented in green. Striped bars represent EPS-stimulated cells. EPS: Electrical pulse stimulation, GOCC: Gene Ontology cellular component, NP: non-predicted, SP+: signal peptide positive, SP-: signal peptide negative, FC: fold change, SEM: standard error of the mean, IL-6: interleukin-6.

Article Snippet: Myotubes were stimulated in serum-free medium with EPS (C-Pace EP, IonOptix LLC) for 6 hours with 2 m pulses at 40 V (HSkMCs) and 11.5 V (C2C12) following the most frequently used established protocols for these cell types ( ).

Techniques: Phospho-proteomics, Western Blot, Multiplex Assay, Quantitative Proteomics