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FACS using DAPI for apoptosis analysis

FACS Immunology Apoptosis

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3 replies to this topic

#1 Nabil Zeidan

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Posted 03 June 2013 - 07:21 PM

Hello,

I am treating cancer cells with a drug and want to analyse cancer cell death. The only problem is that the drug auto-fluoresce a lot, so much that I cannot use conventional viable stains like PI or 7AAD (because sometimes spillover cannot be corrected).
I think using DAPI could solve my problem. However, I am not familiar with DAPI. It is often used to determine cell cycle progress, I think, by histology. However, the use of DAPI in FACS would require a much different protocol, I guess (fixation ect.).

I need help, where could I find a good protocol that uses DAPI for the study of apoptosis by FACS ?

Thank you

#2 Curtis

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Posted 04 June 2013 - 07:26 AM

With PI you need to fix too, so how do you stain with PI? I don't understand.

#3 Tabaluga

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Posted 04 June 2013 - 08:14 AM

Maybe by that he means that he thinks a different kind of fixation might be used...
I've no experience with DAPI in FACS, but did you see this paper ? http://jcb.rupress.o.../171/3/559.full

Il dort. Quoique le sort fût pour lui bien étrange,
Il vivait. Il mourut quand il n'eut plus son ange;
La chose simplement d'elle-même arriva,
Comme la nuit se fait lorsque le jour s'en va.

 


#4 Zee

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Posted 25 June 2013 - 11:11 AM

I don't know anything about using DAPI by flow. I wonder if it is detectable?

However, there are additional alternatives. Try Hoescht 33342 from invitrogen. It fluoresces in the UV range, which I assume DAPI will too, but I know it works by flow. You do have to check that the equipment you are using has the correct filters and laser, though.

Edited by rdvelvet02, 25 June 2013 - 11:22 AM.






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