Human skeletal myoblasts (Lonza Group Ltd, Anaheim, CA) were cultured in complete growth media (Lonza). Cells (6 × 105 per well in a six-well plate) were cultured for 24 h, and serum starved for 4 h before treated with myostatin, GDF-11, or activin A at 4 nM in presence of 40 nM of REGN1033 or ActRIIB-hFc for 30 min. Cells were lysed in NP-40 buffer containing 1 % NP-40, 100 mM KCL, 20 mM Tris/HCL (pH 7.6), 1 mM EGTA, and 1 mM NaF with various protease and phosphatase inhibitors and cleared by centrifugation. Soluble fractions were separated in 4–20 % Invitrogen Precast Tris-Glycine gels using SDS-PAGE followed by transfer to polyvinylidene fluoride (PVDF) membranes. Total levels of Smad2/3 and phosphorylated Smad2 were determined with rabbit anti-Smad2/3 and phospho-Smad2 (Ser465/467) antibodies (Cell Signaling Technology, Danvers, MA).
Complete growth media
Manufactured by Lonza
Complete growth media is a laboratory product designed to provide all the essential nutrients and components required for the cultivation and growth of various cell types in vitro. It is a pre-formulated, optimized solution that supports the optimal proliferation and maintenance of cell cultures.
Automatically generated - may contain errors
2 protocols using complete growth media
1
Characterization of Myostatin Signaling
2
Intracellular and Extracellular cGMP Measurement in Renal Proximal Tubule Cells
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Human renal proximal tubule cells (RPTEC; cat# CC‐2553) and complete growth media (cat# CC‐3190) were purchased from Lonza (Allendale, NJ). The cyclic GMP (cGMP) EIA kit was purchased from GE Healthcare (Piscataway, NJ; cat# RPN‐226). The HTRF cGMP Assay kit (cat#62GM2PEC) was purchased from Cisbio (Bedford, MA). 3‐Isobutyl‐1‐methylxanthine (IBMX, cat# I7018) was purchased from Sigma (St. Louis, MO). Cells were cultured at 37°C in a 5% CO2 incubator following the vendor protocol. Cells were seeded in 96‐well plates at 20 000 cells/well in 0.1 mL complete media overnight. The next day, the media was replaced with fresh complete media plus 0.5 mmol/L IBMX and the cells were treated for 48 hours or as indicated in Figure 2 . At the end of the treatment, the cells were harvested and the supernatant collected. A 100‐μL aliquot of Lysis Reagent 1 provided in the cGMP EIA kit, plus 0.5 mmol/L IBMX, was added to each well to lyse cells for the intracellular cGMP measurement. The protocol from the EIA kit was followed to determine intracellular cGMP levels. The levels of secreted cGMP in the supernatant were determined by the HTRF method following the kit instructions.
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