Hello,
I was trying to do phalloidin staining last week using U87 cells (Adherent cells) to see actin fibers. These cells are generally in an elongated shape, but all my cells tend to get rounded during the staining process. I am not sure what the problem is. Here is my staining protocol.
I gelatin coat my cover slips and grow U87 cells on that. Then I wash with 1X PBS +10% FBS once. After that, I fix cells with 4% PFA for 20 minutes at RT. (After this step my cells get rounded up). Then again wash cells 2 times with 1X PBS +10% FBS. Then Permeabilize cells using 0.05% Triton solution for 2 minutes. Wash 1 time using 1X PBS + 10% FBS. Stain cells with Phalloidin-iFluor 488 Reagent (ab176753) for 30 minutes at RT. Again wash with 1X PBS + 10% FBS. Finally, do the DAPI staining and mounting slides with ProLong Diamond Antifade Reagent.
I would really appreciate it if anyone can help me with this.
Thank you.
Try warming up the PBS for the first wash. The cells are getting shocked by the quick change in temp and retracting.
Yes as Chris mentioned and yourself mentioned, the rounding up of the cells happened after the fix. You may want to pre-warm to 37c whatever wash buffer you are using as well as your 4% PFA. Try that first and observe if your cells round up after that. If they do, then there is still some more troubleshooting that needs to occur.
Christopher B O'Connell and Aaron Dhanda Thank you very much for your responses, I will try these ways as well.
Fixation SHOULD crosslink the proteins and prevent cell morphology changes, as well as crosslinking cell proteins to the gelatin matrix.
Make sure the formaldehyde is freshly-made (or freshly opened, in the case of sealed aliquots). It could be that if it is old it is not properly fixing the cells.
- Badges
- Science topic
- Similar topics
- Engineering
- Modeling