Hello everyone,
I am trying to do surface staining of a protein of interest in adherent cells for analysis in FACS. However, I am not getting what I am expecting, and I am wondering if something in my cell preparation is going wrong. Specifically, if I'm correctly treating the cells with the drugs. I would appreciate it if you could take a look at my current protocol and give some feedback if you think I'm missing/doing something wrong.
Here's my protocol so far:
1. Treat the cells for the desired time with the desired drug on the T-25 plates (cells have grown to the desired density for flow (~80% conf)).
2.Trypsinize the cells and spin down (at 4C) to remove trypsin ( I have transferred them to Eppendorf tubes)
3. Wash once with PBS (at4C)
4. Wash with cold PBS (at 4 C)
5. Add the primary antibody to each of the tubes and incubate on ice for 30 minutes.
6. Wash twice with Flow cytometry staining buffer from eBioscience (https://www.thermofisher.com/order/catalog/product/00-4222-26)
7. Add 3.7% PFA to fix cells at room temperature for 10 mins
8. Spin down to remove excess PFA
9. Wash with FC staining buffer
10. Resuspend in FC buffer for storage until FACS experiment. Store at 4C covering them with foil.
It is important to note that, starting from step 4, I have placed my samples on ice the whole time to prevent endocytosis.
Please let me know if you have any suggestions.
Thank you in advance,
Valeria
Since you are staining for a surface bound protein marker, trypsin could cleave your protein of interest during the cell harvesting step. So I suggest you use a gentler enzyme like accutase for flow cytometry purposes or just 10mM EDTA solution in DPBS (works for some cell lines). Also the antibody mentioned in the 5th step contains a fluorescence probe right? As I cannot see a secondary antibody staining step. Also the PFA fix is not necessary if you are doing the analysis immediately after staining and are not storing the samples.
Hi Ashutosh Acharya ,
Thanks for the suggestion! I'll try using the EDTA solution, but I'm not sure it will work for some of the cell lines I use since they strongly adhere to the dishes.
Yes, the antibody already contains a fluorescent probe.
Sometimes I don't have immediate access to the flow cytometry facility, so that is why I usually fix my samples, but whenever I do, I'll try not to fix them.
Thank you so much for your suggestions!
- Badges
- Science topic
- Similar topics
- Medicine
- Epi
- Age Groups
- Children