Isolation of granuloma macrophages from the livers of Infected mice

Equipment and reagents

• Tissue homogenizer (Biospec Inc.)

• Rocking platform

• 250 ^m sterile nylon mesh (PCG Scientific)

• Dissecting board

• Pathogen-infected mice

• Collagenase type I (Sigma Chemical Company)

• Hypotonic lysis buffer (Tris-NH4C1) {see Protocol 11)

Protocol 19 continued

Method

1 Sacrifice the mice by cervical dislocation or C02 asphyxiation.

2 Place each mouse on a dissecting board ventral-side up.

3 Clean the abdomen with 70% ethanol.

4 Make a longitudinal incision exposing the peritoneal cavity.

5 Remove the liver lobes with sterile forceps. Use only livers that exhibit multiple small whitish granulomas.

6 Collect the livers in a 50 ml tube containing enough MEM to cover all organs,

7 Homogenize the tissue using a low to medium setting on the tissue homogenizer for 4-7 min or until tissues appear to be completely in suspension.

8 Place the homogenate in a 50 ml tube and allow the granulomas to sediment for 5 min.

9 Decant the supernatant and resuspend the pellet in the same volume of MEM as initially used.

10 Repeat sedimentation procedure three more times.

11 Resuspend the final pellet in MEM containing 1000 U/ml collagenase at a volume equal to that of the granulomas.

12 Incubate the cells for 30 min at 37 °C on a rocking platform.

13 Stand the tube upright and allow the undigested granulomas to sediment.

14 Decant the supernatant through nylon mesh into a new 50 ml tube.

15 Wash the cells three times with MEM by centrifugation for 10 min at 300 g at room temperature.

16 Lyse the red blood cells by hypotonic shock using Tris-NH4C1,

17 Wash the cells again with MEM.

18 Resuspend the cells in MEM plus 10% FCS.

The resulting population is 30-45% macrophages, which can be further purified by adherence (see Chapter 2) or by negative selection using antibodies against Thy-1, heat stable antigen, and granulocytes in the presence of 10% rabbit complement for 30 min at 37°C (51, 53). In the negative selection procedure, macrophages can then be obtained by separation or a self-forming Percoll gradient (see Protocol 10), which results in a 95% pure macrophage population.

6.2 Tumour-associated macrophages

The same levels of tissue dissociation discussed in Section 5.1 apply to tumour-associated macrophages. Isolation of these cells may require more trial and error since each tumour is different and incubation with enzymes may be longer or shorter. Histology is an important prerequisite to isolation with respect to expected yields and contaminating cell types. The protocols described below are a good starting place and have been used successfully in mammary tumours

(54, 55). A more extensive review of enzymatic tissue dissociation is reported by Russell et al (55). These methods were optimized for murine tumours. However, similar methods can be used for isolation of macrophages from human tumours (55, 56). Protocol 20 describes the isolation of tumour macrophages by mechanical dissociation; Protocol 21 describes their isolation by enzymatic digestion.

Protocol 20

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