Help for TUNEL method for apoptosis

Immunobiology Research Institute, Siena unutmaz at IRIS02.BIOCINE.IT
Thu Apr 20 02:24:51 EST 1995


	
	Recently we have been trying to setup the TUNEL method to detect apoptosis 
of mouse thymocytes in suspension, according to the published method by Kishimoto H. 
et. al. 1995 J. Exp. Med. 181:649. But we have encountered two major problems, we 
would appreciate very much any help or suggestions for the following:

1) Our first problem is the recovery of cells, expecially after refixation with 1% 
paraformaldehyde following initial fixation with 70% ethanol. At this step we lose 
95% of our starting cells.

So far we have tried different spinning conditions but the yield never improved. Is 
there a particular speed and/or temperature to centrifuge the cells after the  
paraformaldehyde treatment?

2) Our second problem is the level of fluoresence intensity after staining the cells 
with biotin-dUTP followed by PE-conjugated streptavidin. 

The incubation temperature with streptavidin is not mentioned in the above protocol. 
(We only tried rt). Is it possible that it could also be a problem of  
permeabilization of the cells (We just followed the protocol for this point).

Thanks in advance.
	
Derya, Sandra, Oretta

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Derya Unutmaz, Sandra Nuti, Oretta Finco
Immunobiology Research Institute, 
Siena, Italy.
E-mail: Unutmaz at iris02.biocine.it
Fax: +39-577-24 35 64	
	



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