Isolation of macrophages from dissociation

Equipment and reagents

• Sterile 250 ji.m nylon mesh screens (PGC2 Scientific)

• Sterile rubber stoppers

• Sterile forceps and scissors

• Table-top centrifuge solid tumours (mechanical

• 75 mm2 sterile Petri dishes

• Pasteur pipettes

• 19% Nycodenz (density 1.1 g/ml) (Accurate Chemical Co) (see Protocol 13)

Method

1 Cut the tumour into 2-3 mm3 pieces of tissue.

2 Pass the tissue pieces through a sterile screen mesh into a Petri dish by pressing on tumour sections with a sterile rubber stopper.

3 Collect the cell suspension in a fresh 75 mm2 Petri dish containing 2-3 ml of ice-cold RPMI1640 with 10% FCS.

4 Layer the resulting cell suspension onto a Nycodenz density gradient.

6 Aspirate the cells at the interface with a Pasteur pipette.

7 Wash the cells twice with RPMI 1640 and centrifuge at 300 g for 10 min.

8 Resuspend the final pellet in RPMI 1640 with 10% FCS.

9 Purify the macrophage population by adherence (see Chapter 2).

Protocol 21 j

Isolation of tumour-associated macrophages (enzymatic

method)

Equipment and reagents

• Sterile forceps and curved scissors

* Digestion medium: DMEM, 50 U/ml colla-

• 125 ml Erlenmeyer flask

genase type II (Worthington Biochemial

• Sterile magnetic stir bar

Corp.), 10 U/ml DNase (Calbiochem)

• Sterile gauze

• HB SS plus 10% FCS (Gibco BRL) (see

• DM EM (Gibco BRL)

Protocol 5)

Protocol 21 continued

Method

1 Trim the tumour of any fat, grossly necrotic, or haemorrhagic pieces with sterile scissors.

2 Mince the tumour into approx. 1 mm3 pieces with curved scissors.

3 Digest the tumour mince in a 125 ml Erlenmeyer flask in 50 ml digestion media. Stir with a magnetic stir bar for 20 min at room temperature.

4 Stop stirring and allow the tumour suspension to settle for 2-3 min,

5 Carefully collect the supernatant and wash it immediately in cold HBSS with 10% FCS to neutralize any residual enzyme.

6 Repeat steps 3-5 three more times.

7 Pool the supernatants and filter them through sterile gauze to remove clumps.

This cell suspension can then be further purified by centrifugal elutriation (see Chapter 2).

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