Equipment and reagents
• Sterile scissors and forceps
• 60 p.m sterile gauze (Fisher Scientific)
• 15 ml polypropylene tubes
• Pasteur pipettes
• Dissociation buffer: GBSS, 0,2% pronase (Gibco BRL), 0.8 |u,g/ml DNase (Boehringer Mannheim)
1 Mince the biopsy tissue into small pieces (1-2 mm3 in size) in GBSS.
2 Incubate the liver fragments in 75 ml dissociation buffer with continuous stirring at 37 °C for 30 min. During incubation period, check and correct the pH to 7.3-7.5 with 1 M NaOH, as needed.
3 Filter the resulting cell suspension through 60 ^m gauze. Complete tissue dissociation may require reincubation of unsuspended tissue for another 15 min in dissociation buffer.
4 Spin the cells at 300 g for 10 min to pellet them.
5 Wash the pellet twice in 50 ml buffer II,
6 Resuspend the pellet in 5 ml buffer II.
7 Layer the resulting cell suspension over 16% Nycodenz.
16% Nycodenz (Accurate Chemical Co) in isotonic buffer: 0.75% NaCl, 5 raM Tris-HCl pH 7.5, 3 mM KC1, 0.3 mM CaNa2EDTA
Protocol 13 continued
8 Spirt the gradient at 600 g for 20 min at 4°C in a 15 ml polypropylene tube.
9 Collect the interface cells with a Pasteur pipette.
10 Wash the cells in 10 ml GBSS.
11 Spin the cells at 300 g for 10 min.
12 Resuspend the pellet in buffer II.
13 Enrich the human Kupffer cells by counterilow centrifugal elutriation (see Chapter 2). iii. Osteoclasts
Osteoclasts are the macrophage-like cells of the bone. These cells are specialized for bone resorption. Osteoclasts are often multinucleated as a result of frequent cell fusion and the majority of cells stain positively for tartrate-resistant acid phosphatase (TRAP) after three days in culture (37). Although osteoclasts exist in low numbers in the bone, the protocol described below outlines steps to enrich for these cells (38). Further purification of these cells is possible by micromanipulation of cells under phase-contrast microscopy (38) and is not presented here. Techniques similar to those described can be used to enrich for osteoclasts in other mammalian species including humans (37). From 1 cm3 of starting material, approximately 1.2 x 106 cells can be harvested (37). Protocol 14 describes the isolation of murine osteoclasts.
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