lymphocyte culture - problem in isolating lymphocytes from spleen culture (Feb/02/2007 )

Dear friends,

I am new with spleen cell culture work. I want to isolate lymphocytes from spleen but can't do it using Histopaque. I am not able to get any layer after centrifuging the cell suspension with Histopaque.
I have done the culture twice by the following protocol. Isolation of spleen - Dissociation and filtering of cells - Washing at 1000 rpm for 10 min twice in 5 ml of fluid - Adding Histopaque half in volume of cell suspension - Centrifugation at 1200 rpm for 40 min .
Once i did the whole procedure with RPMI 1640 medium complete along with FCS. In the second trial, i used PBS in the beginning and used complete RPMI medium before the Histopaque step.
Both the times, i was not able to get the separation of lymphocytes.
I am also attaching my protocol, so that u can have a look at it, and might suggest my mistake.

Thanks,

Varun

Hi,
We work with B cell cultures isolated from spleen. We use CD43 magnetic beads (Miltenyi) for the isolation and we get great results with them. It´s quite expensive but it´s fast and worth.
We also use cold PBS 1X - 1% FCS for the whole procedure, not RPMI.
If you want to take a look to our protocol I can send it to you.
Good luck.