IEL Strip Medium (DTT/EDTA)

Dillon Corvino

DTT/EDTA epithelial stripping medium for releasing intraepithelial lymphocytes from the small intestine.
Published

September 18, 2026

Protocol ID: BUF-IEL-STRIP Version: v1.0 Author: Dillon Corvino Date: September 18, 2026

Purpose

Used for the DTT/EDTA epithelial stripping step in intraepithelial lymphocyte (IEL) isolation. DTT reduces disulfide bonds in mucus and epithelial junctions; EDTA chelates calcium, destabilising E-cadherin-based junctions — together with mechanical agitation, this releases IELs from the epithelial layer.

Linked protocols

  • MUS-IEL-ISO

Working buffer composition

20 mL per mouse, made from 3% Medium (BUF-IEL-3PCT) with DTT and EDTA added fresh.

DTT stock: 14.5 mg/mL. MW of DTT = 154.25 g/mol -> 14.5 mg/mL = 94 mM stock. For 1 mM final in 20 mL: add 213 uL DTT stock.

EDTA stock: 500 mM. For 5 mM final in 20 mL: add 200 uL EDTA stock.

Component Final concentration Volume for 20 mL
3% Medium Base 19.6 mL
DTT (14.5 mg/mL stock, ~94 mM) 1 mM 213 uL
EDTA (500 mM stock) 5 mM 200 uL
Warning

Prepare fresh and pre-warm to 37 °C immediately before use. DTT is unstable in solution and loses activity over time. Do not store.

Preparation

A. Preparation of stock (if applicable)

Prepare 3% Medium (BUF-IEL-3PCT) in advance — this forms the base. DTT and EDTA stocks may be pre-made and stored (DTT stock at -20 °C; EDTA stock at room temperature), but must only be added to the base medium immediately before use.

B. Preparation of working buffer

  1. Add DTT and EDTA stock to 3% Medium per the table above.
  2. Mix gently by inversion.
  3. Pre-warm to 37 °C in a water bath immediately before use.

Storage and stability

  • Do not store — prepare fresh and use the same day.
  • DTT loses activity in solution over time; do not prepare in advance.

Reagent details

Component Supplier Cat# Notes
DTT (dithiothreitol) [TBD] [TBD] Prepare 14.5 mg/mL (~94 mM) stock; add fresh
EDTA (0.5 M stock) [TBD] [TBD]

Safety (brief)

  • DTT is a reducing agent with a strong odour; handle in a well-ventilated area and avoid inhalation.
  • Standard laboratory PPE otherwise.

Version history

Version Date Author Change summary
v1.0 2026-09-18 Dillon Corvino Split out of prot_mus_iel_isolation.qmd into its own buffer document (buffer/protocol architecture redesign) — no change to composition.