I am trying to start culture with smooth muscle cells isolated form human pulmonary arteries, I would like to know what is the best culture medium to use and the percentage of fetal bovine serum?
Article Serum Can Overcome Contact Inhibition in Confluent Human Pul...
Remove the cryopreserved vial of HPASMC from the liquid nitrogen storage tank using proper protection for your eyes and hands.
Turn the vial cap a quarter turn to release any liquid nitrogen that may be trapped in the threads, then re-tighten the cap.
Thaw the cells quickly by placing the lower half of the vial in a 37°C water bath and watch the vial closely during the thawing process.
Remove the vial from the water bath when only a small amount of ice is left in the vial. Do not let cells thaw completely.
Decontaminate the vial exterior with 70% alcohol in a sterile Biological Safety Cabinet.
Remove the vial cap carefully. Do not touch the rim of the cap or the vial with your hands to avoid contamination.
Resuspend the cells in the vial by gently pipetting the cells 5 times with a 2 ml pipette. Be careful not to pipette too vigorously as to cause foaming.
Pipette the cell suspension (1ml) from the vial into the T-75 flask (SIAL0641) containing 15 ml of Smooth Muscle Cell Growth Medium (311-500).
Cap the flask and rock gently to evenly distribute the cells.
Place the T-75 flask (SIAL0641) in a 37oC, 5% CO2 humidified incubator. Loosen the cap to allow gas exchange. For best results, do not disturb the culture for 24 hours after inoculation.
Change to fresh Smooth Muscle Cell Growth Medium (311-500) after 24 hours or overnight to remove all traces of DMSO.
Change Smooth Muscle Cell Growth Medium (311-500) every other day until the cells reach 60% confluency.
Double the Smooth Muscle Cell Growth Medium (311-500) volume when the culture is >60% confluent or for weekend feedings.
Subculture the cells when the HPASMC culture reaches 80% confluency.
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