Epithelial cells are susceptible to fibroblast contamination, the extent of which can be determined by double immunofluorescence staining using antibodies specific for the intermediate fibroblast subunit vimentin (as a marker of fibroblasts) and keratin (as a marker of epithelial cells). Methods on how to reduce contamination have been described previously; another method used to reduce the number of fibroblasts sprouting from a population of adherent epithelial cells was trypsinization using different concentrations. This experiment was obtained from the Cell Lab Guide (previous volume) by Peitang Huang.

Operation method

Reduces contamination during epithelial cell culture

Principle

Epithelial cells are susceptible to fibroblast contamination, the extent of which can be determined by double immunofluorescence staining with antibodies specific for the intermediate fibroblast subunit vimentin (as a marker of fibroblasts) and keratin (as a marker of epithelial cells). Methods for reducing contamination have been described previously; another method used to reduce the number of fibroblasts in the adherent epithelial cell population is trypsinization at different concentrations.

Move

course of events

1) Wash the culture once with HBSS containing 0.53 mmol/LEDTA,0.05% trypsin.

2) Add the same concentration of freshly prepared trypsin to the culture and leave for 4 minutes at room temperature.

3) Wash the culture by blowing the cells up and down with a pipette to aspirate the trypsin.
The rounded fibroblasts are gradually removed during the blowing process, leaving only the epithelial cells.

4) Add fresh culture medium to inactivate the residual trypsin.

Caveat

Note of caution.If necessary, the above culture medium can be supplemented with a fungicidal reagent such as 2.5ug/ml amphotericin B. Unfortunately, fungicides may prevent cell growth and should not be used whenever possible.

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