Loading soluble antigens into macrophages by osmotic lysis of pinosomes

Equipment and reagents

• Centrifuge equipped with Sorval H1000B rotor (Falcon)

• 50 ml polypropylene tubes

• C02 incubator

• Antigen (e.g. ovalbumin, Sigma) for which class I-restricted T cells are available

Method

1 Dissolve soluble antigen (e.g. ovalbumin), in hypertonic medium.

2 Make dilutions of antigen in this medium over a range from 0.1-10 mg/ml.

3 Obtain macrophages from desired source (see Chapter 1).

4 Seed 5-10 x 106 macrophages at 106/ml in RPME to several conical polypropylene tubes.

5 Centrifuge to pellet cells (1200 r.p.m., 10 min, RT).

6 Remove medium by vacuum aspiration.

7 Resuspend each cell pellet gently in 1 ml of pre-warmed (37°C) hypertonic medium containing antigen at a specific concentration.

8 Incubate macrophages with antigen at 37°C, 5% C02 for 10 min.

9 Dilute the cell suspension with 15 ml of hypotonic medium.

10 Incubate an additional 3-5 min at 37°C.

11 Wash cells three times by centrifugation in excess norma! strength RPMI, to remove exogenous antigen.

12 Use cells in functional assays as desired.

• Hypertonic medium: 0.5 M sucrose, 10% polyethylene glycol 1450 (Sigma), 10 mM Hepes in RPMI medium

• Hypotonic medium: 60% (v/v) RPMI medium in dH^O

Protocol 6 outlines the most successful protocol wc have developed for assessing the stimulation of class I-restricted responses by BMM0. The comparison between "osmotically loaded" macrophages and control "untreated" macrophages pulsed with soluble antigen and with specific peptide epitopes allows the relative efficiency of these routes of antigen uptake to be evaluated.

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