How to culture C2C12 cells?
How to culture C2C12 cells?
C2C12 cells are cultured in growth medium in collagen-coated 150 cm2 polystyrene flasks at 37 °C in humidified atmosphere of 5% CO2. CRITICAL STEP: To avoid premature myoblast differentiation, cells are kept at very low confluence (~ 40%) and propagated before they reach higher confluency.
How to thaw C2C12 cells?
1. Thaw a 1-ml aliquot of cells as quickly as possible in water bath at 37°C. Transfer cells to 24 mL warm media in a 50 mL conical tube.
What cells form myotubes?
Primary myotubes are developed from embryonic myoblasts and can differentiate into both slow (type I) and fast fiber types (type II). This differentiation occurs before the motor nerve axons have contacted the newly formed muscle.
What are L6 myotubes?
L6 cells, originally derived from rat skeletal muscle, propagate as mononucleated myoblasts but can differentiate into multinucleated primary myotubes. The myotubes express several proteins typical of skeletal muscle including the GLUT4 glucose transporter.
How big are c2c12 cells?
At the time of the biophysical studies, mean myotube diameter was 12 microns (range 5-25 microns), and mean length was 290 microns (range 130-520 microns).
What is myogenic differentiation?
Myogenic differentiation proceeds through irreversible cell cycle arrest of precursor cells (myoblasts), followed by a gradual increase in expression of muscle function genes, leading to fusion of myoblasts into multinucleate myofibers in the animal.
How do you revive ATCC culture?
Upon receipt of frozen cultures, immediately revive cultures by thawing and subsequently transferring cultures to an appropriate growth medium. If this is not possible, store frozen vials in liquid nitrogen vapor (below -130°C).
How do you unfreeze a cell culture?
Video: Thawing cells
- Remove the cryovial containing the frozen cells from liquid nitrogen storage and immediately place it into a 37°C water bath.
- Quickly thaw the cells (< 1 minute) by gently swirling the vial in the 37°C water bath until there is just a small bit of ice left in the vial.
What is myogenic index?
The myoblast fusion, or myogenic index, is defined as the number of nuclei residing in cells containing three or more nuclei divided by the total number of nuclei in a given image [20].
What to do with a C2C12 cell line?
Here are some basic methods for dealing with C2C12 cells: Proliferation of C2C12 cells Heat growth media (GM – DMEM (25mM glucose), 20% fetal bovine serum, and penicillin/streptomycin) to 37 degrees Celsius
How often to split C2C12 cells in DMEM?
Split cells normally 1/10 or 1/20 into 10% FBS in DMEM with 1x PSG (see Splitting Cells) Try to split at 70-80% confluence. Healthy growing cells will reach confluence every other day after a 2X dilution. Split them as fibroblasts into the final format (12 well plate, 6 well plate etc.)
When to split C2C12 cells in 100 mm dishes?
Cells normally need to be split every other day and we maintain them in 100 mm dishes, if they are not ready to split, refeed them on the second day. When cells reach 80-90% confluence switch to media (DMEM, 1x PSG with 2% horse serum).
How often do healthy C2C12 cells reach confluence?
Healthy growing cells will reach confluence every other day after a 2X dilution. Split them as fibroblasts into the final format (12 well plate, 6 well plate etc.)