Adherent Cells Protocol
Cells in culture are either a form cultured in suspension, such as blood cells, or cells such as fibroblasts requiring a surface to which they can adhere. Some of the surfaces may require coating in different extracellular matrices depending on cell type and study. All cell cultures require to be performed are aseptic conditions.
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Optimum Environment
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The majority of cell types can be cultured in Dulbecco's modified Eagle's medium, (DMEM) or Roswell Park Memorial Institute (RPMI) with the addition of 10 percent fetal calf serum, 2 milimoles of the amino acid glutamine and antibiotics, for example, 100 units penicillin and 0.1 milligram per milliliter streptomycin. Cells are maintained in their media in a humidified incubator at 37 degrees Celsius and 5 percent carbon dioxide.
Splitting
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Cells should be checked daily for infection, death or growth. Once the tissue culture flask is 80 percent coated in cells, the cells should be divided to maintain the cells' health and the log phase of growth. Media are removed and the cells are washed in phosphate buffered saline. Adherent cells require the addition of trypsin and ethylenediaminetetraacetic acid (EDTA) to remove cell adhesion proteins. Gentle agitation should remove the cells. Media needs to be added to neutralize the trypsin/EDTA solution.
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Subculturing
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The diluted cells and tryspin/EDTA solution can be centrifuged to pellet and wash the cells. The cells can be re-suspended in media and diluted and replated into new flasks. Each time the cells are split the cells passage number is increased by one.
Media Change
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Media should be changed twice a week by removing the old media and replacing with new, warmed media.
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