Hi

You may be able to fix the cells (i.e. kill them) with the readout you want intact for FACS analysis This should satisfy your HSE colleagues. I am with them on adhering to BS2 (and above) if thats how the cells are categorised. A relatively light risk assessment produced in discussion with them and your FACS team is probably the best way forward.

Hi,

As mentioned above, fixing (killing) the cells will be the way to downgrade them to the safety level 1. Also lysates (proteins/RNA/DNA) from the cells may be considered as level 1. But (at least in Germany) it is also necessary to make a special report/document where you explain why the sample can be downgraded to the safety level 1, signed by the responsible people and the safety officer of your institution.

Best,

Michail

As for your quesions about the virus particles... in general, immortalized cell lines that were done with viruses are still considered to have the capability of generating viral particles. Unless ATCC certifies that the cell line is virus free, always assume that viral particles can be present. (There were several news on virus contimination of other cell lines because of this. I can't remember the details, but I seem to remember that ATCC has to send out notices for this years ago.)

For the specific one that you are using, the risk is very low that there would be any viral particles that can be generated due to the type of virus used.

As for the analysis... what are you doing? (we do this a lot for BSL-3 bacteria cell culture work that needed to be analyzed in the BSL-2 or BSL-1.) For flow, since the cells are fixed anyway after staining, the samples are now BSL-1 compliant. For supernatants, you will have to do the entire ELISA in the BSL-2, and you can take out the plates once you add the perioxidase, which at that point is also BSL-1 compliant. For DNA/RNA, once you add the stablizer (such as a phenol based one) that lysis the cells, the samples are also fine.

Thank you all very much! I usually don't fix my cells before FACS since I am staining for extracellular markers. But I guess it should be doable.

So I guess that there is a small risk of the creation of virus particles, hence a small risk that my leukocytes will be infected during co-culture. How would you test this? By PCR?

Hi Johanne,

No, you should stain for the markers before the fixing (you are correct that extracellular markers should be stained before fixing). What I meant was that the cells are fixed before you run them through the flow machine.

You should be able to do the staining in the BSL-2. If you don't want to bring you antibody stocks in there, I would just prepare the stock solution in the BSL-1 and bring that into the BSL-2. They should have everything else you need in the BSL-2.

I won't worry about the potential viral infection during co-culture at this stage. The cell line has publications associated with it, do a quick search to see if anyone tested for viral particles. If they did, and they didn't see any, I would just reference them in your studies as to why you don't need to repeat that particular analysis.