Equipment and reagents
• Tenbroeck tissue homogenizer (Bellco)
• 50 ml polypropylene tubes
• Sterile gauze 2 in x 2 in (Fisher Scientific)
• Sterile forceps and scissors
• MP medium: RPMI 1640. 2 ^M l-glutamine, 10 ng/ml garamycin, 1% tiypticase soy broth, 10% heat-inactivated newborn calf serum (Gibco BRL)
1 Cut the splenic tissue (10-20 g) into pieces in a Petri dish containing 5-10 ml MP medium.
2 Load the contents of the Petn dish into a Tenbroeck tissue homogenizer and homogenize the sample with eight to ten strokes.
• Collagenase type VIII (Sigma Chemical Co)
• Tris-NOtCl: add 90 ml of 0,16 M NH4C1 to 10 ml of 0.17 M Tris pH 7.65, and adjust to pH 7,2 with HC1
• Heat-inactivated newborn calf serum (Gibco BRL)
• Bovine pancreatic DNase I (Sigma Chemical Co)
Protocol 11 continued
3 Decant the suspension and stroma into a 50 ml polypropylene tube.
4 Wash the suspension twice by centrifugation for 10 min at 400 g in MP medium at 4°C.
5 Resuspend the cells to 3 x 10B cells/mi in MP medium.
6 Add collagenase at 260 U/ml of cell suspension.
7 incubate at 37°C, 5% C02 for 30 min with occasional agitation.
8 Pellet the cells by centrifugation for 10 min at 400 g at 4°C.
9 Resuspend the cells in 1 ml Tris-NH4C1/0.1 ml packed cells.
10 Leave the suspension at room temperature for 2 min.
11 Underlay the cells with 5 ml of newborn calf serum.
12 Centrifuge the cells at 400 g for 10 min at 4°C.
13 Wash the cells twice as described in step 4.
14 Resuspend the cell pellet to 3 X 10s cells/ml in MP medium containing 20 (ig/ml bovine pancreatic DNase I.
15 Incubate the suspension for 30 min at 37 °C, 5% C02 with occasional agitation.
16 Dispense the stromal fragments by gently pipetting.
17 Dilute the cell suspension in MP medium (enough for easy filtering) and filter the cells through a sterile gauze pad into a 50 ml polypropylene tube.
18 Centrifuge the cells at 400 g for 10 min at 4 °C.
19 Resuspend the cells in 5 ml MP medium containing 20 (j.g/ml fresh DNase I for further purification by adherence of countercurrent centrifugal elutriation (see Chapter 2).
i. Human splenic macrophages
Human monocyte/macrophage populations are most often studied using peripheral blood due to availability and ease of isolation. However, the human spleen is an abundant source of macrophages and these cells can be obtained by a combination of mechanical and enzymatic dissociation (33). This method yields a cell suspension containing approximately 13% macrophages (most of the splenic macrophage population}. Macrophages can be further isolated by plastic adherence (see Chapter 2) but for highly enriched cells without selective loss, countercurrent centrifugal elutriation is recommended (see Chapter 2).
ii. Kupffer cells
The liver is comprised of parenchymal cells (hepatocytes) and non-parenchymal cells. The majority of non-parenchymal cells are sinusoidal cells. Of this subset, 60% are endothelial cells and 40% are Kupffer cells. Hepatocytes can be separated from non-parenchymal cells due to their sensitivity to collagenase (34-36). Kupffer cells can then be separated from endothelial cells by centrifugal elutriation (see Chapter 2). Approximately 1.8 x 10s cells per gram of wet liver tissue can be obtained from mice. These cells are 98% viable and 95% pure Kupffer cells (34), Protocol 12 describes the isolation ofKupffer cells from pathogen-free mice.
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