Approximately 50 x macrophages can be obtained from 25 g of tissue. Skin-associated macrophages Langerhans cells are macrophage-like cells that reside in the epidermis. Protocol 18 describes the preparation of Langerhans cells from the skin of pathogen-free mice 45, Try to keep the skin in one piece.
Do not use the tail or lower legs. Decant the supernatant fluid. Keep skins and cells on ice unless noted otherwise.
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. These include tuberculosis, sarcoidosis, cat-scratch disease, leprosy, brucellosis, and schistosomiasis 49, A granuloma is identified by its distinctive pattern of inflammatory reaction in which aggregates of epithelial-tike macrophages are surrounded by lymphocytes and a few plasma cells.
Older granulomas also display an outer layer of fibroblasts and connective tissue. In some cases, fused macrophages, giant cells, are also found Protocol 19 describes the isolation of granuloma macrophages 51 from livers or lungs 52 of infected mice. Use only livers that exhibit multiple small whitish granulomas. 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.
A more extensive review of enzymatic tissue dissociation is reported by Russell et til. These methods were optimized for murine tumours. However, similar methods can be used for isolation of macrophages from human tumours 55, Protocol 20 describes the isolation of tumour macrophages by mechanical dissociation; Protocol 21 describes their isolation by enzymatic digestion. Stir with a magnetic stir bar for 20 min at room temperature.
References 1. Rutherford, M. Biol, 53, , 2. Morahan, P.
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Macrophages: A Practical Approach
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Macrophages A Practical Approach Practical Approach Series
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