 |
 |
 |
 |
>
>
> We have just started using a CD69 and CD71 based lymphocyte function
> assay (based on some work I've previously done and the BDIS paper in Cytometry
> 20:127-133 (1995)).
> Generally I am seeking a group opinion on whether this method is valid
> to replace the tritiated Thymidine method, and, what ways can it be improved
> to make it more viable an alternative.
>
> The method uses ficolled lymphocytes (1million) stimulated for 4 hours
> with PWM, Con A, CD2, PHA, SEB, Candida A., PMA as a maximal control and an
> unstimulated control. These are then FACSed 4 hours using CD3/CD69 for
> activation and again at 16hours with CD3/CD71 for proliferation.
>
>
> My Questions:
>
> 1. Is there a need for an additional proliferation marker (eg. CD25) or is
> CD71 alone acceptable.
>
> 2. Would more co-stimulatory mitogens (eg. CD2 and 28) be more relevant
> clinically to test activation/function than mitogens like PWM and ConA.
>
> 3. Would DNA profiles be of any real advantage in this case.
My only comment on this would be that BrDU incorporation would probably
give the closest correlation with tritiated thymidine, and would also
give you a DNA profile to compare results with if you wished.
> Brett Teale
> Queensland Institute of Medical Research/Dept.of Immunology,
> Royal Brisbane Hospital
> Queensland
> Australia
>
> brettT@qimr.edu.au
Eric Miller, ICRF,Edinburgh UK
|
|
|
|