IEL Isolation Protocol
- RPMI + 1mM DTT
- RPMI + 5% FCS + 1mM EDTA
- Ca-, Mg-free Hanks Balanced Salt Solution (HBSS) + 25mM HEPES + 1mM DTT + 1mM EDTA, pH 7.6
- 90% Percoll = 9 parts percoll + 1 part 10x PBS + 1mM EDTA
- 70% Percoll
- Facs buffer + 1mM EDTA
- Nylon wool columns x3 (~60mg/10ml syringe)
- Remove whole small intestine and dissect away any attached connective tissue. Place in petri dish containing ice-cold RPMI.
- Place small intestine on paper towelling pre-wetted with RPMI. Dissect out Peyers’ patches and any remaining connective tissue.
- Cut longitudinally, being careful not to scrape mucus away with intestinal contents. Cut tissue into 0.5-1cm lengths.
- Wash gently in 2 changes of ice-cold RPMI (alternative – can flush lumenal contents out by slowly passing through RPMI before any cutting). Transfer to 50ml Falcon tube containing RPMI + 1mM DTT.
- Incubate in 25ml RPMI + 1mM DTT for 15 min at room temperature to remove mucus. Centrifuge at 1500 rpm for 5 min at RT to pellet tissue and cells, and discard s/n.
- Shake in shaking/orbital incubator (140/min) in pre-warmed Ca, Mg-free HBSS + 25mM HEPES + 1mM DTT + 1mM EDTA (25ml/mouse) at 37°C for 30 minutes.
- Shake vigorously by hand (or vortex) for 30 seconds. Separate isolated cells from remaining tissue by passing cells over 3 successive pre-wetted (with FACS EDTA) loose nylon wool columns to remove mucus, the majority of epithelial cells and debris – to give greater purity of isolated IEL. Centrifuge cells at 1500 rpm for 5 min at RT.
- Resuspend pellet well in 5ml of RPMI + 5% FCS + 1mM EDTA and add 4ml of 90% percoll (= 40%). Try to avoid bubbling. Overlay the 9ml onto 3ml of 70% percoll (2.33ml of 90% + 0.67ml of 1x PBS) in 15ml clear falcon tube. Centrifuge for 20 minutes at 1500 rpm at 20 ° C.
- Remove IEC from top of the upper 40% percoll layer. Collect from 40-70% percoll interface and transfer to 10ml tube.
- Wash x2 in 10ml FACS EDTA and pellet cells to remove percoll. Resuspend pellet and count cells with haemocytometer.
Yield is usually 1-2 x 10^7/mouse, 50-80% of which are IEL.