Re: CD34 CD133

Hi Irina,
if you are getting different results with every other CD34 conjugate  
compared to CD34PE, there is something very wrong with either the  
antibody conjugates you have used, or perhaps you are not deploying  
them properly in your 'ISHAGE-like' procedure.
I am not sure what you mean by 'ISHAGE-like'; either you are using  
the ISHAGE protocols properly, as written, or you are using something  
else!
We have tested multiple antibody clones (581, 8G12, Birma K3 [all  
class III reagents]) and a variety of conjugates thereof, on the same  
samples on the same day and we generate essentially identical results.
We generate the same data using class II reagents (QBEnd10) labelled  
with PE on these very same samples. The FITC conjugates of class II  
reagents fail to accurately derive the 'correct answer' because of  
'negative charge issues' associated with FITC conjugates of this  
antibody.  Other than this one well characterised problem, things  
'just work', as long as the assay is properly deployed.
When we compare the data obtained with Beckman Coulter reagent sets,  
with BD Biosystems reagents sets, and Dako reagent sets, we obtain  
the same results using the standardised ISHAGE protocols. Even 'bad'  
PECy5 conjugates generate the same data as 'clean' ones if you deploy  
the assay properly, because non-specifically stained cells are 'gated  
out' by the sequential boolean gating strategy employed at the heart  
of the protocol.
If you have any data files you would like to share with us, perhaps  
we could help identify the problems you are experiencing.

best,
Rob Sutherland
University Health Network
Toronto

On 21-Dec-07, at 3:23 PM, Irina Grigorieva PhD wrote:

>
> Jos,
>
> When speaking of quantitative analysis of stem cells, I would not  
> substitute CD34-PE with any other fluorochrome. We tried to useCD34- 
> APC in our ISHAGE-like procedures ( I am not even saying PerCP) and  
> saw a difference right away.
>
> If you plan just to characterize the population of interest, we use  
> CD34-PerCP-Cy5.5: we like it just fine and we have data. And PE-Cy7  
> may be a good choice, though I have no data to support this statement.
>
> Irina
> Irina Grigorieva, PhD
> Director, Flow Cytometry Laboratory
> Northside Hospital, Atlanta, GA
> (404)- 851-6541
> e-mail: irina.grigorieva@northside.com
>
>
>
> From: Jos Bosch [mailto:j.a.bosch@bham.ac.uk]
> Sent: Thursday, December 20, 2007 10:11 AM
> To: Cytometry Mailing List
> Subject: CD34 CD133
>
> Dear Flowers,
>
>
>
> Am setting up a panel on a 6-color FACScanto to characterize stem  
> cells in peripheral blood, and running into the usual planning  
> problem on what fluorochromes to assign to particular antibodies.  
> The main hassle is caused by the fact that CD133 (miltenyi) and KDR  
> (R&D) are only available in PE and APC, so have to work around these.
>
>
>
> To make a long story short, there are two options I seem to be left  
> with:
>
> 1)	  Substitute CD34 PE (clone 8G12, BD) for PerCP.
>
> Arseniev (1999) concluded PerCP was not really bad, but not really  
> great either. What is your experience?? Was unable find any  
> systematic comparison in the literature that looked at PE-Cy7 or  
> APC-Cy7. Does anyone have good validation data?
>
> 2)	  Alternatively, I can buy CD133 biotin and label it with FITC.
>
> Does anyone have good experience with that approach? What can I  
> expect with regard to signal strength, background, and prep hassle?
>
>
>
> Your advise is very much appreciated!
>
>
>
> Kind wishes,
>
>
>
> Jos
>
>
>
> Jos A. Bosch, PhD
>
> Lecturer in Exercise and Behavioural Immunology
>
> School of Sport and Exercise Sciences
>
> University of Birmingham
>
> Birmingham  B15 2TT
>
> United Kingdom
>
>
>
> T: (+44) 0121 4148105
>
> F: (+44) 0121 4144121
>
> http://www.sportex.bham.ac.uk/staff/boschja
>
>
>
>
>
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