alternative to facs?

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alternative to facs?

Postby faariwasi » Dec 17 2014 11:01 pm

I am starting a new project and I need to express a protein in HIV infected Jurkat cells and then use these cells for further experiments. I cannot sort the cells because we do not have a sorting facility (FACS) in our PC3 and these infected cells cannot leave the PC3 lab. So I was thinking about expressing my protein of interest and at the same time a cell surface receptor not normally express by Jurkats in order to isolate expressing cells with beads binding to this expressed receptor.
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Re: alternative to facs?

Postby tluebke » Aug 06 2015 7:18 am

What do you want to do afterwards with these cells? Do you want to test them directly or let them grow in culture? Is your protein expressed by some cells only? Otherwise a real sorting from a Jurkat bulk culture is not neccessary, I would say.
Depending on what you want to do, you can do the following things:
- Aditionally express GFP and estimate the number of GFP positive cells via fluorescence microscopy. By analyzing you also can calculate percentages.
- Use a resistance marker (e.g. puromycin) and kill all cells which were not targeted by your virus.
- Do a dilution in a 96 well plate (1 cell/well to get single cells in a U or V-bottom plate) and let them regrow. You can test the over expression via western blots afterwards.

Maybe this helps you.
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