RE: Nuclei abosolute counts

Hi,
I'm no expert in running clinical samples, but I have been running a lot of absolute count assays on L5178Y mouse lymphoma cells with invitrogen's beads CountBright (UV to 635 nm excitation and 385-800 nm emission) on a FC500 MPL.
 
I have also used BD's flow count beads, but they tend to aggregate a lot more even with vigorous vortexing.
 
I'm wondering why you would want to have an absolute count of these nuclei? The result you will get is: number of nuclei/ml. Does that make any sense to your assay? 
 
I'm just curious...
 
 
 
Best regards,

Mr.Morten Hellmers Fjordholt 
H. Lundbeck A/S 
Molecular Toxicology, dept. 855 
Non-Clinical Safety Research 
Ottiliavej 9 
DK-2500 Copenhagen Valby 
tel:   +45 3643 4036 
cel:  +45 3083 4036 
fax:  +45 3643 8313 

 


________________________________

	From: Roberts Joanna [mailto:joanna.roberts@epfl.ch] 
	Sent: Monday, July 10, 2006 9:27 AM
	To: Cytometry Mailing List
	Subject: Nuclei abosolute counts
	
	

	Hello flow people !

	 

	I would like to use a 'count' bead such as 'flow count' or similar to get an absolute count of Hoeschst stained breast cancer cell-line nuclei. 

	 

	If anyone is using count beads to get absolute counts by flow for nuclei preparations, it would be great to know what works well for you.  

	 

	If anyone sells count beads that could work, I would like to hear about it also.

	 

	I think I will need a count bead that can be excited by the violet laser to produce a signal around 450nm. This is because I may need to set a detection threshold using the hoechst signal (with a 450/50 emission filter and excitation off a violet laser) to see the nuclei if I cannot get a clear scatter signal to set a threshold on. 

	 

	I will use a CyAn ADP for this and I think one can only trigger on a single parameter here. Is this correct?

	 

	Any comments or advice or suggestions very much appreciated. 

	 

	Thanks so much for all the helpful responses that come out of this list, 

	 

	Best, 

	Joanna 

	 

	 

	 

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