Hello,
I am always cautious on prevention of cell cross contamination.
What I express as 'Cross contamination' here is not the mix of cells
of totally different types, but same type of cells treated differently.
I often transfect 'control siRNA' to some HUVEC samples(dishes, wells, etc) and transfect 'siRNA of gene of interest' to other HUVEC samples(dishes, wells, etc.). After dropping siRNA molecules,
I handle all these samples with same suction tip in clean bench because
changing suction tip takes too much time for cell works.
There seems to be no critical problem so far when considering research outcomes, but I am concerned about the cross contamination of cells
because of use of one suction tip, especially in trypsinization.
I am considering about situation when cells or other components from one specific sample attach to suction tip when suction tip touches bottom of culture monolayer and
move to other samples. (But only so small part of monolayer touched)
I want to hear opinions from others whether such concern is
critical or can be simply neglected.
Thank you!
I'm always using the same glass pipette for aspirating the medium of all samples (I guess that's what you mean?) and I never had any problems with cross contamination. But I think it depends on your application. When you are doing siRNA treatment, you transfect your cells transiently I guess? So then it shouldn't be a problem. In my case, the inhibition is never completely, more 60-90%, so I guess if there are very few cells from another well, this doesn't make a difference!
I hope this helps you a little bit!
Dear Ariane Eberhardt,
Thank you for your answer!
Of course, your reply is very helpful.
So you mean that cells or other components causing
cross contamination due to attachment to
suction tip are so minute to affect research outcomes?
I appreciate you again!
Contamination from one sample to another may be a minor problem. But I'd use a different tip for different cells. Just as you probably do with tips/syringe for cell lysis. You can aspire some sterile water between aspiration of different cells if you consider a waste of material to change the tip.
Dear Noa Martin, thank you for your support!
If you don't mind, can i ask what you expressed as
'Different cells' means same cells treated differently
or cells of different types? So you think cross contamination between same cells treated differently is neglectible during steps including harvest of trypsinized cells?
I appreciate you again!
Ok, I am not completely sure what you mean. Do you mean that you suck the medium/PBS out with the same pipette/tip, or do you mean that you actually collect cells with the same tip?meaning, after trypsinization and neutralization, you use the same tip to collect cells, but you just put them in different tubes?. If the case is the first (you just take out the medium), it is ok, the number of cells in the medium that will end up in another wells will not change at all your results. If you mean the second (using the same tip to actually collect the cells) I would be more concerned. The number of cells that stick to the tip/pippete can be, I think, relatively high. Perheps it doesn´t bias your results, but this is definitively not optimal.
Dear Victor Sarachaga,
Thank you for sharing your opinion!
Oh, I mean that I use same suction tip for
removing trypsin, PBS , media, etc. I never use same tip
when collecting cells treated differently in different tubes.
I am just a bit concerned about cells or other components attached to suction tip
that can transfer to cells treated differently especially in
trypsinizations and also in other steps(ex)From siControl to experimental)
because suction tip touches very small parts of cell monolayers
when suctions are almost finished in each samples.
If you have any comments, I would be deeply appreciated.
Dear Lee,
When I say different cells I mean differently-treated cells.
The optimal is to use a different tip.
Upon use of trypsin, you will have dettached cells. Usually, I recover the cells with the trypsin and add some media with FCS or BSA to avoid cell lysis. It is not usual to aspire trypsin and then use another solution to recover cells, but this may depend on your cell typo and protocol, of course. Anyway, at this point, you can mix a lot of cells.
Indeed, if you change the tip from control to treated cells, then you will close the tube where you collect your cells and this also helps to avoid pouring the cells into the wrong tube.
However, if you mix 100-1,000 cells with 1,000,000 I do not think this will make the difference. But it is not optimal. Indeed, if you trypsinize a lot of dishes at a time, this will affect the latest cells you recover. This can make a difference.
In my opinion it does not create any significant difference so far outcome is concerned. For example when i do pi staining i do not change tips for adding RNase or Propidium iodide in cells treated with different targets or scrambled shRNA.
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