Standard Operating Procedure

Depletion of Langerhans Cells in vivo

Hazards:

Diphtheria toxin (DT) is a potent lethal toxin in humans. The minimum lethal dose is 100ng/kg (6-10 micrograms). The toxin is harmful is inhaled, injected, or absorbed through the skin or eyes. The toxin has no effect if administered orally, as it is unstable at acid pH. If inadvertent skin pricking should occur, encourage bleeding and perform vigorous flushing of the area with copious amounts of water and/or saline. If i.v. or i.m. injection should occur, seek a physician's attention immediately.

Protection:

Individuals performing this procedure should have a recent (within last 5 years) Diptheria/Tetanus booster vaccine (available from Boyton Health Services).

DO NOT WORK WITH THE TOXIN IN THE DRIED STATE. Work only with reconstituted material. Work in a well-ventilated area and avoid inhalation of the product. Wear protective gloves, lab coat, and eye wear when handling the material. Avoid contact with open wounds. Wash thoroughly any area of the body that comes into contact with the toxin.

Waste:

Contaminated pipettes, lab ware, etc should be disposed of as hazardous waste (i.e. put in an appropriately labeled red biohazard bag, and autoclaved).

Spill clean-up: Remove any contaminated clothing and put in red autoclavable biohazard bag.

Notify other workers in the area of the spill and control traffic through area.

Wear shoe covers if spill is on floor. Put on gloves and cover spill area with paper towels.

Pour bleach over towels from edges of spill to center, be careful not to splatter. Decontaminate all objects in spill area. Allow 20-30 minutes of contact time. Pick up any sharps, including broken glass, with forceps and place in sharps container. Wipe area with disinfectant and clean towels, mop if spill on floor. Remove gloves and foot covers before leaving area of the spill, put in biohazard bag, and wash hands. Autoclave waste.

Procedure:

Preparation of toxin: Work in the hood in 6-250 NHH. Reconstitute a single vial of diphtheria toxin powder (List Labs #150) with 1ml of sterile dH20 (using a syringe and needle to plunge through the septa of the vial). Each vial, when reconstituted contains 1.0 mg/ml of diphtheria toxin in 0.01 M Tris and 0.001 M Na2EDTA, pH 7.5. Aliquot into 40 vials (25 ug each) and store at –20oC. Label freezer box well “Diphtheria Toxin – HAZARDOUS”.

Prepare mice for injection: Label cage cards “Diphtheria Toxin – Hazardous”. If working in a procedure room in MCB, be sure to bring appropriate protective equipment and biohazard bags with you.

To treat mice: Thaw a single aliquot and dilute 1:100 or 1:1000 in PBS (final concentration 10 ug/ml or 1 ug/ml). Load into a tuberculin syringe (27g needle) and inject 100ul i.p. into mice (1ug or 100ng respectively). Do not recap needle, dispose of directly into sharps container.

There is minimal risk to animal caretakers as the amount of unmetabolized DT excreted by the injected mice is likely to be extremely low. Bedding and carcasses can be disposed of as usual.