Immunofluorescence staining of spheroid cells - (Apr/13/2007 )
Do anyone has experience in staining spheroid cells? Since the spheroids wont attch firmly to culture substrates, I lose them after the whole staining procedure.I am specifically looking for the expression of a tyrosine kinase receptor expression on these spheroids? Expecting reply from experienced hands
Spheroid cells? As in cells got spherical shape?
- What cell line are you doing exp with?
- Are they suspension cells (e.g. lymphocyte...) or adhesion cells?
- Is the spherical shape their natural morphology, and do you need it to stay that way for your exp?
- How did you do IF?
- If they are suspension cells, the method for IF could be different.
- You can make the cells spread out more (if you don't really need them to stay in round globes) by using coated coverslip, e.g fibronectin, collagen.... There are many types.
- If the main concern is about losing cells, the coated coverslip can help there, and/or alternatively:
+ Cells mostly lost (washed away) BEFORE fixing. So to prevent this, you need to pay more attention to the steps BEFORE and IN the FIXATION period.
+ So, be gentle and careful with cells when you take them out of the incubator, when you wash and fix them. Better don't use shaker. Put the washing buffer in GENTLY (tip the plate, put the pipette's tip on the well's wall and let the liquid run down SLOWLY), slowly tip the plate back an forth a few time then discard the washing buffer. Putting the fixing buffer in the same way. After this, cells should stick well, you don't have to worry anymore.
+ NOTE that the two above steps are for adhesion cells or when you intend to fix cells on coverslip. If you are working with suspension cells and/or want cells to stay suspended through out staining process, that will need different procedure, as I said before.
Hope that help
can you recommend a protocol for suspension cells?
You want to fix them on coverslip?