How to get rid of contamination in stable transfected cells - (May/05/2005 )
I am working on stable cell lines. Couple of days ago, my hela cells were infected. is there anyway i can get rid of the bacteria or yeast? Otherwise, i have to strat with transfection and selection again, which is time consuming.
Usually when an infection occurs I just start again, except if using stable cell lines, which obviously require considerable work to get back to square one.
In the past I have used a product called Fungizone in my media you treat the cells for a couple days with an almost toxic amount and then gradually reduce the dose. It will kill off most fungi and bacteria, however, if cells are heavily contaminated they won't survive. Can't remember which company just do an internet search.
When an irreplaceable culture becomes contaminated, researchers may attempt to eliminate or control the contamination.
First, determine if the contamination is bacteria, fungus, mycoplasma, or yeast. Isolate the contaminated culture from other cell lines. Clean incubators and laminar flow hoods with a laboratory disinfectant, and check HEPA filters. If the cell line or culture is not one-of-a-kind, it is best to throw it away and start over with new cells. However, you may try to save the culture with the following procedure. Antibiotics and antimycotics at high concentrations can be toxic to some cell lines. Therefore, perform a dose response test to determine the level at which an antibiotic or antimycotic becomes toxic. This is particularly important when using an antimycotic such as Fungizone or an antibiotic such as tylosin.
The following is a suggested procedure for determining toxicity levels and decontaminating cultures.
(1) Dissociate, count, and dilute the cells in antibiotic-free media. Dilute the cells to the concentration used for regular cell passage.
(2) Dispense the cell suspension into a multiwell culture plate or several small flasks. Add the antibiotic of choice to each well in a range of concentrations. For example, we suggest the following concentrations for Fungizone: 0.25, 0.50, 1.0, 2.0, 4.0, and 8.0 µg/ml.
(3) Observe the cells daily for signs of toxicity such as sloughing, appearance of vacuoles, decrease in confluency, and rounding.
(4) When the toxic antibiotic level has been determined, culture the cells for two to three passages using the antibiotic at a concentration one to two-fold lower than the toxic concentration.
(5) Culture the cells for one passage in antibiotic-free media.
(6) Repeat step 4.
(7) Culture the cells in antibiotic-free media for four to six passages to determine if the contamination has been eliminated.
Thank you veteran. I will have a go tomorrow! Thank you again for your help!