I am trying to stain chicken bone marrow derived dendritic cells (BMDCs) with Hemagglutinin (HA) fused CD11c single chain fragment variable (scFv) antibody for flow cytometry. The staining with CD11c scFv on its own is fine. When I tried staining with HA-CD11c I realised that lot of cells were stained. I doubted this was due to the non specific binding of HA to BMDCs. As a control I have stained BMDCs with HA on its own, without CD11c scFv antibody and I get similar staining. Hence, I am not being able to compare the binding of my targeted HA -CD11c scFv with the untargeted counterpart.
My constructs HA-CD11c scFv and HA have V5 tag and my secondary antibody is against V5 tag (anti V5 FITC).
I don't know how to reduce the non specific binding of HA antigen to BMDCs. Do I increase the blocking time or BSA concentration in blocking buffer?
The protocol I use for Flow cytometry labelling of chicken BMDC is as follows:
-Resuspend 5x10^5 cells per well in FACS buffer (PBS+0.5%BSA) for 10-15 minutes.
-Add primary antibody (HA-CD11c scFv or HA) and incubate at 4C for 1.5-2 hours.
-Wash 2 times with FACS buffer
-Add secondary antibody goat anti V5 FITC for 45 min and incubate at 4C.
Fix cells and read the pates in a flow cytometer.
I would be really grateful if anyone could give me suggestions on how to reduce the non specific binding of HA antigen to chicken BMDCs?
Thanks
Angita
I see a few critical points in your protocol, which in my view are very important to rectify. Although I don’t have much experience in working with Chicken bone marrow-derived DCs, I have a good experience in Mouse bone marrow-derived DCs and FACs analysis. Hope these points can help you rectify your problem those are as follows:
Try to use some blocking antibodies instead of increasing BSA in FACS buffer before staining the cell surface.
Incubation time with primary antibodies is very long if possible try to optimize between15 min to 30 min. There is a high chance that the antibodies are taken up by cells through endocytosis.
The incubation time for Secondary antibodies also needs to be reduced.
How are you fixing the cells, over fixation can enhance the autofluorescence, and if possible just avoid the fixation step.
All the very best
Hello,
First do the counting of your samples and add antibody accordingly. One more important aspect is low dose or minimal use of BSA. In addition, try to altwr the incubation period and do all ur processess using 4 degree condition.
Regards
Hi Prashant,
I am fixing the cells using paraformaldehyde for 20 minutes.
Also, if I use blocking antibodies against hemagglutinin would it not bind to the secondary antibody? The blocking antibodies would be anti mouse hemaglutinin and secondarty anti mouse FITC?
Regards,
Angita
There are many blocking buffers available in the market, You can also find antibodies raised in different species if possible and make your own blocking buffer. Tagged primary antibodies for chicken would have solved your problem if you can find. Anyways there are other options you need to optimize conditions as I wrote earlier and use 5% FBS (In your case chicken serum may work) in your FACS buffer that will work better than BSA.
What is the percentage of Paraformaldehyde, it should be 4% and incubation time is again a problem. Either skip this step or fix in 2-4% PFA for 5-10 min. My suggestion would be to compare your results with or without fixation, you will be able to decide which one is better.
Don't forget to use isotope control in your experiment.
Hope This can work.
All the very best for your experiment
- Badges
- Science topic