HT29, EDTA or thrypsin? - (Jun/15/2007 )
I am now doing on testing a compound on HT-29. As HT-29 is anchorage dependent so i would need to use either EDTA or trypsin. But i am not sure using which would be better. Would anyone with experience in this helping me? plus, as i am new in cell culture, i am not able to differentiate yeast, fungal, bacterial contamination and cell debris that usually presents inside the culture. Can anyone gives me some tips or recommend me any useful website? thanks alot
in our lab we use Trypsin containing 0.25% EDTA for our HT-29.
protocol online itself has a lot of informative methods/protocols/tips to read. when i just started to do cell culture work, i refer to protocol online cell culture technique a lot. very useful.
as for contamination, when you look under microscope, if you see something like trees, and have tree-like branches... then it should be fungus contamination. if you see some black dot moving under microscope, then it should be bact contamination.
normally, if contamonation happens, your media (which contain phenol red as pH indicator) will change colour and the media become cloudy. just like you culture E.coli in broth... the broth will become cloudy. as some bact can doubled itself in 20 minutes, bact contaminated culture will become VERY cloudy the next day. you'll easily notice the difference. if your media is clear and you actually can look through it, that means your culture is OK, even though sometimes you can see some debris in it.
just to be safe, DO NOT USE the cells once you notice there is something not right with your cells, eg, funny morphology, funny shape things inside, hard to trypsinesed/takes long time to attach (healthy cells should attach and detach easily), slow growing........
finally, do not passage your cells toooooooooo many times. your cells might change its characteristic after passage too many times. after certain period, thaw another vial from early passage to do your experiment. i always take note of the passage no. of cells and after, maybe like, 100-200 passage/2-3 months, i'll take another vial. it is hard to know how many passage is too many passage, but definately not forever. if your experiment carried out using cells between 80-100 passage, always use cells within that passage no. for the rest of your experiment.