Peritoneal cells

6.1 Peritoneal cells

Peritoneal-derived immune cells are essentially made up of macrophages and lymphocytes (predominantly CD5 -B1 lymphocytes) and may be prepared by washing the peritoneal cavity of normal mice or guinea-pigs. For many purposes it is possible to elicit larger numbers of macro- phages by producing a local inflammatory response, e.g. with starch, sodium trioleat, paraffin oil, etc. However it is important to remember that macrophages elicited in this wash will contain engulfed particles and will not be normal.

6.1.1 Isolation of normal peritoneal macrophages

MATERIALS Mice or guinea-pigs Tissue culture medium containing 10% fetal bovine serum 1% w/v neutral red in saline

METHOD

1 Kill the mouse or guinea-pig by cervical dislocation.

2 Inject 8.0 ml of tissue culture medium into the mouse’s peritoneal cavity (80 ml for guinea-pigs).

3 Knead the abdomen gently to bring the cells into suspension.

4 Open the abdominal skin to expose the peritoneum.

5 Using a syringe with a 21-gauge needle, push the needle into the peritoneum, roll the mouse on its side and aspirate the medium. Alternatively, insert the needle into the peritoneal cavity towards the xiphisternum with the needle bevel directed downwards. The ventral peritoneal wall can be raised slightly with the syringe needle and the internal organs tend to settle, this reduces the possibility of fat bodies blocking the needle during withdrawal of the fluid.

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6.1PERITONEAL CELLS

6 Collect the peritoneal cells by centrifugation (150 g for 10 min at room temperature) using siliconized glassware or plastic.

7 Estimate the number of phagocytes using a haemocytometer by the uptake of a 1% neutral red solution.

TECHNICAL NOTES • A normal mouse will yield 5 × 10 6 peritoneal exudate cells, up to 50% of which will be lymphocytes.

• Although more of the peritoneal infusion can be collected by using a pipette after opening the peritoneum, care is needed if the cells are intended for sterile use. • Most of the phagocytic cells will be macrophages, but there is likely to be a small amount of contamination with neutrophils.

6.1.2 Eliciting peritoneal exudate cells

MATERIALS Mice or guinea-pigs

1% starch in 0.14 M saline Tissue culture medium 1% w/v neutral red in saline

METHOD

1 Inject 2 ml of starch suspension into the peritoneal cavity of the mouse (25 ml for guinea-pigs).

2 Kill the animals after 3 days.

3 Inject 2–5 ml of tissue culture medium into the peritoneal cavity and gently press the abdomen to bring the cells into suspension (80 ml for guinea-pigs).

4 Open the abdominal skin of the animal and hold up the centre of the peritoneum with forceps.

5 Make a small hole in the peritoneum and remove the medium with a pipette.

6 Finally, open the animal fully and suck out all the medium. To handle these peritoneal exudate cells you must use either siliconized glassware or plastic.

7 Estimate the number of phagocytes by the uptake of a 1% neutral red solution (haemocytometer count).

The exudate should contain approximately 75% phagocytes and 25% other cells; this may be confirmed by non-specific esterase staining. Although more cells are obtained after starch treat- ment, remember that some of them will be activated as the starch induces a mild inflammatory response.

6.1.3 Non-specific esterase staining of macrophages

Peritoneal macrophages stain more strongly for non-specific esterase activity (particularly if they have been elicited by an inflammatory stimulus) than macrophages and monocytes elsewhere in the body. The latter types of cells are still easily distinguished by this staining reaction.

C H A P T E R 6: Isolation of cells

MATERIALS AND EQUIPMENT Source of macrophages or monocytes

1 g pararosaniline

10 M HCl Phosphate buffers Acetone Formaldehyde solution Sodium nitrite, 4% w/v in water, freshly prepared

0.1 M sodium hydroxide

a naphthyl acetate Methyl green dye, 0.4% w/v in water DePeX artificial mountant Equipment for smear preparation

Preparation in advance

1 Prepare a stock solution of the stain by mixing 1 g pararosaniline with 5 ml concentrated HCl (10 m) and 20 ml distilled water.

2 Heat to 70°C, allow to cool to room temperature, filter and store at 4°C in the dark.

METHOD

1 Prepare a smear of the cells on a microscope slide, either manually, or using a cytocentrifuge (see Section 11.6).

2 Fix the cells for 30 s at 4°C in 30 ml of 0.1 M phosphate buffer, pH 6.6, mixed with 45 ml acetone and 25 ml formaldehyde solution.

3 Wash three times with distilled water and air dry.

4 Immediately prior to use, prepare the active diazonium salt, hexazotized pararosaniline by mixing 6 ml of 4% w/v sodium nitrite (freshly prepared) with an equal volume of the pararosaniline stock solution and dilute to 200 ml with 0.067 M phosphate buffer, pH 5.0.

5 Adjust the activated dye solution to pH 5.8 with 0.1 M sodium hydroxide; this is the optimum pH for esterase activity.

6 Dissolve 50 mg α-naphthyl acetate in 2 ml acetone and add to the staining solution in step 5.

7 Incubate the smears in the stain for 4 h at room temperature or 45 min at 37°C, rinse in distilled water and counterstain for 1–2 min in a 0.4% aqueous solution of methyl green dye.

8 Wash the smears in distilled water, air dry and mount with DePeX.