Hi, I'm trying to isolate colonic IEL/LPL following a protocol from Jove “Isolating Lymphocytes from the Mouse Small Intestinal Immune System”(yeah that's weird to use this protocol since it‘s designed for small intestinal IEL/LPL isolation but I can't find a good one for colon) and cannot have a satisfying yield of IEL cell number and viability, although it's quite good when it comes to LPL.

In brief, the washed colon (cut open longitudinally then into ~2cm pieces) would be digested twice in 1mM DTE solution(with 10% FBS) while being stirred at 37 °C and ~220 rpm on a magnetic stirrer for 20 min. After each digestion, I would vortex the pieces for 10 s and filter the supernatant through a 70-µm cell strainer, then combine and pellet the filtered supernatant, use gradient density centrifugation (44/67 percoll) to collect IELs. The remaining pieces would be further digested by EDTA to remove IECs then collagenase to collect LPLs.

As a result, LPL isolation is fine based on the cell number/viability/H&E staining but it doesn't work for IEL. Sometimes I even can't find the pellet after centrifugation although a clear but thin white layer can be seen in percoll centrifugation, and trypan blue staining always show a