Colonic cell isolation was performed as previously published with a few modifications (20 (link)). Briefly, colons were removed, cleaned of mesentery and feces, and opened up longitudinally. They were then transversely cut into small pieces of ~1 cm in length, and intraepithelial and LP fractions were extracted following the manufacturer’s protocol (LP dissociation kit, a MACSmix tube rotator, Miltenyi Biotec). Following two rounds of predigestion solution using the MACSmix tube rotator, the IEL fraction was generated. The remaining tissue was washed to remove the EDTA, and supernatant was again pooled with the IEL fraction (third wash). The colon pieces were then incubated in digestion solution (containing enzymes) at 37°C and gently shaken as mentioned using the MACSmix tube rotator. Finally, tissue pieces were disrupted using the m_intestine_01 program of the gentleMACS dissociator and passed first through a 100-μm filter and then through a 70-μm filter to obtain the LP fraction.
For the small intestinal IEL fraction, a similar method was followed. For mesenteric lymph node (MLN), lymph nodes were carefully isolated and made free of extra fat and then physically mashed gently using the plunger of a 5-ml syringe while passing through a 70-μm filter to obtain single-cell suspensions.
For the small intestinal IEL fraction, a similar method was followed. For mesenteric lymph node (MLN), lymph nodes were carefully isolated and made free of extra fat and then physically mashed gently using the plunger of a 5-ml syringe while passing through a 70-μm filter to obtain single-cell suspensions.
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