experience with RAG -/- mice - Leukocyte isolation from RAG-/- lymph nodes (May/26/2007 )

Hi,

does anyone reading this have experience with leukocyte isolation from lymph nodes in RAG KO mice? I have been trying to isolate leukocytes from Balb/c RAG KO mice of varying ages (appr. 8-12 wks) and was rarely successful independent of what isolation media +/- supplements I used. I can see tiny lymph node like structures during the isolation procedure but on the FACS they do not stain leukocyte markers and are all small and highly granular cells (FSC, SCC) what I interpreted as being either RBC, dead cells or any other cell (fatty tissue) but unfortunately not leukocytes.

Do younger RAGs have bigger LN? Do B6 RAGs have better LN than Balb/c (for whatever reason)? Are there any secrets about not loosing these apparently highly susceptible cells during isolation and staining?

Any comment is highly appreciated. Thank you very much in advance.

Hi,

why you could not use the spleen for isolating leukos? I think you will be more successful. I only have experience with isolation of leukos from lymphnodes from normal C57/Bl6 mice and they have lymphnodes of a nice sice to isolate. I always take the para-aortal (renal nodes) or the inguinal ones they are of a good size I think, and easy to find.

I hope that this information is helpful for you.

Do you need a particular type of Leukocytes or any of them?

No experience with LNs. How much you need and to what level of purity?