title: 1354
organism: Homo sapiens
description: Protocols: Media were changed immediately before stimulation and collected and centrifuged before frozen at -80°C. Cells were washed 3 times in ice-cold PBS, harvested and frozen at -80°C. CON/EPS of media with no mytobes was also collected as a control of the muscle cell effect. EPS medium was fractionated into a large-molecule containing fraction and a small-molecule containing fraction by Amicon Ultra-2 centrifugal filter units with a cut-off at 3 kD (Millipore) according to manufactures protocol. Briefly, 2 ml medium was pipetted into the fractionation tubes and were centrifuged at 4000 g for 1 hour. The two fractions were stored on -80°C until further use. Primary muscle cells were obtained from postmenopausal women (n=6). Definition of postmenopausal was: women with amenorrhea for more than 12 months and levels of FSH > 20 IU/I. Exclusion criteria included; infections for the last 4 weeks, chronic diseases, medicine use including contraceptives, and BMI > 30 kg/m2. For the experimental day, subjects were fasting overnight and had refrained from physical exercise from 24 hours. Skeletal muscle biopsies from the vastus lateralis of the quadriceps femoris were obtained by the needle biopsy method as described by Bergstrom (18) under local anaesthesia with 2 % lidocaine (SAD). Isolation of satellite cells were performed after the following protocol: Connective tissue and fat were removed and muscle tissue was minced with sterile scissors, followed by digestion in 5 mL digestion solution (HAM/F10 media, containing 0.05 % Trypsin-EDTA, 1 mg/ml Collagenase IV, and 5 mg/ml BSA) for 5 minutes at 37°C. Digestion solution containing liberated satellite cells were transferred to 2 ml fetal bovine serum (FBS) on ice to inhibit digestion. Additional 5 ml of digestion solution was added to the muscle tissue followed by incubation at 37°C for 10 minutes. Both digestion solutions were filtered through a 70µm cell strainer and centrifuged at 800 g for 7 minutes. Cells were washed in HAM/F10 and centrifuged again, resuspended in growth media, (F10/HAM containing 20 % FBS, 1 % penicillin/streptavidin (P/S) and 1 % fungizone (FZ), and pre-plated in a 60 mm plate for 3 hours. Unattached cells were then transferred into a culture flask pre-coated with Matrigel and grown at 37°C in a humidified atmosphere of 5 % CO2. Cells were grown, spilt and finally frozen in liquid nitrogen. Cells were thawn and plated in 6-well plates (matrigel coated) at concentration 3*10^4 cells/well in growth media. When plates were 100 % confluent, media were changed to differentiation media 1 (DMEM, low glucose, 10 % FBS, 1 % P/S). After two days, cells had lined up and differentiation was initiated by switching to differentiation media 2 (DMEM, low glucose, 2 % horse serum, 1 % penicillin/streptavidin). Media from well-trained/untrained myotubes were collected and cells harvested at four different time points during myogenesis: 1) Proliferating; 75 % confluent, 2) Committed to differentiation: 100 % confluent and lined up, 3) Early differentiation; two days into differentiation, and 4) Late differentiation; day five of differentiation. Media were changed 24 hours prior to each collection. Fully differentiated myotubes were induced to contract by two different protocols: Caffeine (CAF) and electrical pulse stimulation (EPS). Myotubes were stimulated to contract by 5 mM CAF (Sigma Aldrich) or by EPS at 11 mV, 1Hz, 2 ms (C-Pace EP, Ion Optix) for 6 and 24 hours in DMEM, 1 % horse serum (HS), 1 % P/S. Non-stimulated (CON) myotubes served as controls. RNA was harvested from the muscle cells by the Trizol method (Invitrogen Life Technologies) according to manufacturer's instructions. RNA concentration was determined by a nanodrop spectrophotometer (Therno Fischer Scientific) and cDNA was made by reverse transcription reactions using Reverse Transcriptase kit and random primers (Applied Biosystem) according to manufacturer's protocol. All qPCR amplifications were performed in triplets using SYBR green master mix (Applied Biosystem) and sequence specific primers. The relative expressions of the measured genes were determined after normalization to the endogenous control gapdh. All primer sequences can be specified upon request. RNA was extracted from the cells using the Trizol protocol, as earlier above, and further DNase treated using Ambion Turbo DNase (Applied Biosystems). Following, RNA integrity was measured on a NanoDrop ND-1000 (Thermo Scientific), and only ratios of 260/280 and 260/230 >2.0 were accepted for further processing. RNA integrity was subsequently evaluated using the Agilent RNA 6000 Nano Reagents Part1 kit on an Agilent 2100 Bioanalyzer (both Agilent, Santa Clara, CA, USA) obtaining RIN-values >9.0 for all samples. The RNA samples were prepared according to the manufactures protocol for TruSeq® Stranded Total RNA Ribo-Zero™ Gold from Illumina. The sample preparation was performed at DTU Multi-Assay Core facility (DMAC), at the institute of DTU Bioengineering, Technical University of Denmark). Briefly, 500 ng of total RNA was depleted of ribosomal RNA using ribosomal removal beads. The samples were purified with AMPpure RNA Clean XT beads (Beckman) and fragmented. The fragmented RNA was processed to a doubled stranded cDNA synthesis using random hexamer primers. A single adenylate overhang was introduced to the cDNA library molecules to facilitate the ligation of adaptor sequences. The adaptor ligated DNA was PCR amplified for 15 cycles. The quality and quantity of the DNA library was subsequently assessed on and Agilent Bioanalyzer and Qubit 2.0 (Invirotogen), respectively