Isolation of murine Langerhans cells

Equipment and reagents

• Single edge razor blades

• Sterile gauze

• Autoclaved paper towels

• Autoclaved 15 cm filter paper circles

• Table-top centrifuge

• Pathogen-free mice

• Langerhans cells (LC) culture medium: RPMI1640.10% FCS. 100 fig/ml

• Lymphoprep (Accurate Chemical Co)

• 2.5% trypsin (Boehringer Mannheim)

• DNase (0.5 mg/ml) (Boehringer streptomycin, 100 U/ml penicillin, 1 x

Fungizone, 2 mM L-glutamine, adjust pH to 7.2

Mannheim)

1 Sacrifice the mouse by cervical dislocation or C02 asphyxiation.

2 Clean the mouse thoroughly with sterile PBS.

3 Shave the body on sterile paper towels.

4 Add 18 ml PBS to a 100 x 15 mm Petri dish on ice (two mice/dish).3

5 Place the mouse on a fresh paper towel.

6 Remove and peel apart the ears.

7 Place the skin from the ear area, epidermis-side up, in the Petri dish.

8 Remove the skin from the rest of the mouse. Try to keep the skin in one piece. Do not use the tail or lower legs.

9 Place the skins on filter paper circles moistened with PBS.

10 Clean the skin of any excess fat or major blood vessels.

11 Cut the skin into 4 mm wide slices.

12 Place the skin in Petri dishes with the epidermis-side up.

13 Add 2 ml of 2.5% trypsin to the 18 ml of PBS (final concentration 0.25% trypsin).

15 Remove the epidermal layer and place it in a new Petri dish containing 8,5 ml PBS, 0.5 ml DNase, and 1 ml tiypsin.

17 Add 2 ml FCS to inactivate the tiypsin.

18 Using a 5 ml syringe (no needle), draw and release the epidermal cells until they become a single ceil suspension.

19 Filter the cells through 40 |xm Nitex mesh into a 50 ml tube.

Protocol 18 continued

20 Centrifuge the cells at 300 g for 10 min at 4°C.

21 Decant the supernatant fluid.

22 Wash the pelleted cells two times in RPMI1640 with 10% FCS.b

23 Count the cells and wash them once more.

24 To enrich the epidermal cells for Langerhans cells, layer the cell suspension (2-5 x 107 cells/5 ml} on an equal volume of Lymphoprep and centrifuge at 400 g for 20 min.

Li Keep skins and cells on ice unless noted otherwise. b If the cells clump, add DNase (50 »1/10 ml).

The recovered cell population is 5-15% Langerhans cells. Culture the cells in LC medium at 2 x 106 cells/ml (47). Langerhans cells can be further enriched by depletion of the dendritic epidermal T cells by incubation with Thy-1 antibody plus complement (45), or by flow cytometric sorting (48) (see Chapter 3).

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