may I ask some tips for IEL isolaton? - (Oct/30/2009 )

Hi, dear all, this is my first time to come here. Unfortunately, I met some questions on IEL isolation from mice. Each time, after I follow the current protocol in immunology to isolate IEL, I always found that I cannot receive enough lymphocytes maybe just 2*10^6 lymphocytes per mouse, and the almost pellet is on the top of 40% percoll, which means they are debris and dead cells. I don't know how to avoid IEL tremendous death and may I ask there are very important notice or tips in IEL isolation? Thank you!

I have tried IEL isolation and it is not fun at all. Some of my tips are....1) get all connective tissue and fat off of the intestine as it will catch cells and hold onto them. This will greatly decrease your yield. I cannot remember what the Current Protocol is but we used EDTA to release the epithelial cells and IELs (make sure that you use PBS without Ca and Mg!) The vast majority of the cells will be epithelial cells (dead and alive). I have also tried Lympholyte M for the gradient. I think this is gentler on the cells (Percoll is toxic to cells if you are not washing it off)


Have fun! Better you than me!