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Tissue Culture Protocol (General): Sterile procedure is of the utmost importance when doing tissue culture.

o Discard any pipets, media or solution you feel may have been contaminated. o Inspect your maintenance cultures carefully and if you see contamination, immediately bleach the flask and inform the other people using the incubator to determine if the whole setup should be autoclaved (contamination can spread quickly and ruin other peoples cultures if you are not careful). Fungal contamination is the most easily identified and usually presents as floating islands of fungus in the culture medium. Bacterial contamination is much harder to spot, but presents as small floating particles (often spinning around) that can be seen under the microscope. Yeast contamination is generally the problem if you have cloudy medium with a distinct smell. Mycoplasma and other viral contaminants cannot be easily identified without PCR analysis, but can sometimes be inferred if a cell line looks off and can then be tested for. Be sure to always wear gloves and any other protective equipment you need to feel comfortable (if you would like a lab coat, safety goggles, etc., please let one of us know). Many of these cell lines are of human origin, and while they have been screened for viruses like HIV and Hepatitis, there is always a danger associated with working with them. Protocol (Cell Splitting)1. Take all of your media and Trypsin/EDTA out to warm/thaw at least 30 minutes prior to when you are signed up for the hood. 2. Be sure to spray down the hood with ethanol if you have any doubt about whether it was cleaned after the previous use. 3. Check your cells to ensure that they are at the appropriate confluence before splitting or treating. 4. Aspirate the old medium off the cells (if you are working with multiple cell lines, be sure to get a new pipet before aspirating a different cell line). 5. Wash the cells once with dPBS (semi-adherent cell lines like COLO 205 or H2122 should have this step omitted). 6. Add 5 mL of Trypsin/EDTA (for a T-75 flask) and place the flasks in the incubator for 30 seconds to 5 minutes depending on the cell line (some take considerably longer to detach from the plate). 7. Gently tap the flask against something to help detach the cells from the flask surface. 8. Add 5 mL of complete media to neutralize the trypsin. 9. Spin the cells at 1000xg for 5 minutes (the purpose of this is to remove the trypsin from the suspension before replating. 10. Aspirate the supernatant from the cell pellet. 11. Resuspend the cell pellet in fresh media and add the appropriate dilutions to new flasks for maintenance or treatment.