I infected the 293tt cell with AAV virus to check the expression of my CAR molecule. According to protocols, I will collect the cells and wash in PBS, than i will stain with a antibody (Fab fragment antibody) for flow cytometry. I want to store my cells, and want to run FACS after few days.
I study few protocols, somewhere they said i need to fix it with PBS and when i used than i will need to stain with antibody. however, somewhere i read that i need to wash with PBS, stain with antibody for 30 mints and than wash with PBS to store.
so which is the better option, i could just wash with PBS and store it. or i also need to stain it with Ab and than store it ? and in case, if i need to stain with antibody, than i could stain with it for 30 mints only or i need to stain it with antibody till i used it ????
thanks.
I normally fix the cells for flow cytometry after staining. Then longest time I have stirred the samples was 40 days which did not effect my results. Here is the protocol.
1. Wash the stained cells in pbs with 2% FCS.
2. Add 3% pfa (paraformaldehyde) in pbs solution and incubate at room temperature for 30 min.
3. Wash the cells with pbs after incubation and add pbs and store them in the fridge.
You need to make sure that the sampes are in airtight vials or parrafilm covered plates for long term storage to prevent the drying.
Alternatively, I also tried resuspending stained and washed cells in 1% pfa in pbs and store until use without any wash steps.
The formaldehyde fixation is not compatible with some fluorophores. Check in bd website for the same.
Goodluck
I also fix the cells for flow cytometry after staining. From my experience, the best fixing condition is using 2% PFA at 4ºC for 10 minutes, then wash, centrifuge and resuspended in FACS Buffer. That way cells are stable for up to 3 weeks.
Hope this helps!
Dear , you will find useful information in links below:
www.enzolifesciences.com › Science Center › TechNotes
www.sciencedirect.com/science/article/pii/002217598790319X/pdf?md5...pid...
https://tools.thermofisher.com/.../staining-cell-surface-antigens-for-flow-cytometry.pd...
Thanks Ramachandramouli Budida, Ana Montalban-Arques, mosab and yong for your valuable suggestions.
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