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Facs for BrdU and other staining


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#1 Rnotk

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Posted 29 July 2010 - 08:04 AM

Hi, All

I am trying to use FACS to analyze the co-expression of GFP, BrdU and other transcription factor expression from mouse brain right now,
but our lab is not really experienced with FACS and I dont even know it works or not.

Especially, BrdU staining require denaturation with acid so I am wondering anyone have any comment about this???

We are also considering useing DNAse treatment instead of acid, but still not sure wheather we can keep detectable GFP expression.

Any suggestion help, so please let me know if you know the protocol or any papers you guys can suggest me to read.

Thanks in advance

#2 rkay447

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Posted 29 July 2010 - 01:57 PM

It took me a long (LONG) time to figure out how to do this but I finally found a protocol that works great. Here is my protocol: first label your cells with Brdu as needed. I label with 10uM BrdU for 20 minutes. Wash cells 3 times with PBS and fix in 3.7% paraformaldehyde/PBS for 10-15 mins at room temp. I buy the 16% paraformaldehyde solution from electron microscopy sciences (cat#15710) and dilute with filtered PBS. Wash cells 2-3 times with PBS. Permiablize cells for 10 mins with 0.5% Triton X-100/PBS at RT. Wash 3 times with PBS. Block for at least one hour in 3-5% filtered BSA/PBS. Stain with desired primary and secondary antibodies (everything but the BrdU). If you want DAPI, you need to do it now as well. Wash cells well as the next step will lock in all background!! You now need to fix the cells again before BrdU staining!!! This is absolutely essential. I just re-fix in 3.7% paraformaldehyde for 10-15 mins. Wash 2-3 times. Next denature the DNA and extract histones with 4N HCl/1% Triton X-100 for 10 mins (for one mL: 600uL dd H2O, 10uL Triton X-100 and 400uL HCl). Ok, so it's 1.01mL, close enough. You must make this fresh each time and it's much easier to add the detergent to the water before adding the acid. Try it once the other way and you'll see what I mean. Wash with 0.1% NP-40/PBS. This wash step is critical to prevent huge background with the anti-BrdU. Be sure to change the buffer multiple times and I always let it wash overnight in the max amount of buffer I could get in the wells. Finally stain with anti-BrdU antibody (I really like the mouse clone BU-33 at 1:1000 for one hour) and appropriate secondary washing with the 0.1% NP-40/PBS buffer. There are also primary-conjugated BrdU and GFP antibodies that work well for FACS and prevent you from having to use secondary antibodies which can greatly reduce background.

Roche puts out a kit that claims to be advantageous by using nucleases rather than acid, base or heat to denature DNA but their protocol calls for a fixative with a pH of 2 so this is kinda bogus. Plus the kit never worked well for me.

Edited by rkay447, 29 July 2010 - 02:06 PM.


#3 Rnotk

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Posted 24 August 2010 - 08:33 AM

rkay447

Thank you for the detail reply.
Since I do use cell from animal, so I probably need to modify your protocol a bit,
but I just optimize protocol for SOX2 and GFP, so hopefully incorporating your reply will give good result!!!
Thanks again, and sorry for the late reply

rnotk




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