Measurement of macrophage antitumour activity by the tumour cell counting assay

Equipment and reagents

• Microtest tissue culture plates, 96-well, flat-bottom, polystyrene (Falcon Plastics, Becton Dickinson Lab ware)

• 25-75 cm2 tissue culture flasks or 60-100 mm2 tissue culture plates, polystyrene (Corn ing-Cos tar)

• Sterile plastic pipettes, 1-10 ml (Coming-Cos tar)

• Centrifuge conical tubes, 15-50 ml polystyrene (Falcon Plastics)

• Cell centrifuge

• Standard microscope

• Haemocytometer

• Device for vacuum aspiration

• Repeating dispenser

• Complete medium: RPM1 1640 (Hyclone Laboratories) or D ME M (ICN Biomedicals, Inc.) supplemented with 10% heat-inactivated, endotoxin-free, fetal calf serum (FCS) (Hyclone Laboratories), 2 inM L-glutamine, 100 U/ml penicillin, and 100 ^ig/ml streptomycin (Celbio S.r.l.)

• Hanks' balanced salt solution (HBSS) (Gibco BRL)

• Ca3+-, Mg2" -free Dulbecco's modified phosphate-buffered saline (PBS) pH 7,2-7.4, containing 8.0 g NaCl/litre, 0.20 g KCl/litre, 0.12 g KH2P02/litre, and 0.91 g Na2HP04/litre

• 0.05% (w/v) trypsin, 0.02% EDTA solutions (Hyclone Laboratories)

• 10% (v/v) Giemsa stain solution (BDH Laboratory Supplies)

• Monocyte/macrophage effector cells, appropriately purified (see Chapters 1 and 2)

• Appropriate tumour target cells maintained in complete medium by serial passages on 100 mm tissue culture dishes or in 75 cm2 tissue culture flasks: e.g. the adherent, spontaneously transformed, murine cell line 3T12 of Balb/c origin for murine macrophage activity or the HT29 human colon carcinoma cell line for human monocyte-mediated anti-tumour activity

• Appropriate macrophage activators: e.g. IFN-y (Genentech, Inc.), IL-2 (Roche), or LPS from Escherichia coli (Sigma)

A Preparation of effector cells

1 Resuspend monocytes/macrophages at 4 x 106 cells/ml in complete medium.

2 Plate 0.1 ml of the macrophage cell suspension (containing 4 x 10s cells) into triplicate wells of a 96-well, flat-bottom micro titre tissue culture plate, using a repeating dispenser.

3 Add the appropriate macrophage activators in 0.1 ml of medium to yield a final volume of 0.2 ml/well.

4 Incubate the cells for 18-24 h at 37 °C with activators.

5 Centrifuge the plate at 250-300 g for 5 min.

6 Remove supernatant by vacuum aspiration.

7 Wash the macrophage effector cells in the plate three times with warm medium or HBSS as follows: add 0.2 ml/well of warm HBSS using a repeating dispenser, centrifuge the plate at 250-300 g for 5 min, and aspirate medium.

Protocol X continued

B Preparation of tumour target cells

1 Wash the tumour target cells, cultured in 25-75 cm2 flasks or in 60-100 mm plates until confluence, once with Ca2+-, Mg3+-free PBS and aspirate the medium,

2 Trypsinize the cells by adding 1 ml of 0.05% (w/v) trypsin in 0.02% EDTA solution for about 2-5 min at 37°C.

3 Observe the cells under inverted microscope and, when monolayers show the first signs of disruption, add 10 ml of complete medium to the flask or plate to inhibit the trypsin.

4 Pipette the cell suspensions several times to separate clumps.

5 Wash the cell suspensions twice in 50 ml of medium each time by centrifugation for 5 min at 300 g in 50 ml conical tubes.

6 Resuspend cells at a final concentration of 1 x 10s cells/ml.a

C Cytotoxicity assay

1 Add 0.1 ml of target cell suspension (containing 1 x 104 cells) to wells containing washed monocyte/macrophage monolayers. Tumour cells should also be added to the wells not containing macrophages.

2 Add 0.1 ml of complete medium to each well to yield a final volume of 0.2 ml/well.

3 Incubate plates containing macrophages and effector cells at 37°C in a humidified incubator for 56-72 h.

4 Remove supernatant from all wells by vacuum aspiration.

5 Wash the plates gently once with PBS.

6 Fix the cells with 100% methanol for 5 min.

7 Stain fixed monolayers by incubating for 7 min with 10% Giemsa stain.

8 Quantify the number of tumour cells remaining in the wells by visual counting clear 'plaques'b in three randomly selected fields of the stained monolayer1 at x 430 mag-nification(x 43 objective with x 10 eyepieces).d

9 Net cytotoxicity is calculated from the average of triplicate samples and determined by the following formula:

{1 - (experimental 3T12 cell number per well/total 3T12 cell number per well} x 100 where the experimental 3T12 cells are tumour cells cultured with macrophages, and total 3T12 cells are tumour cells cultured alone without macrophages.

aThe concentration of the target cells should be adjusted to achieve the desired effector-to-target cells ratio.

b Tumour cells alone continue to proliferate and form a completely confluent monolayer that stains uniformly dark. Activated macrophages destroy tumour cells, inducing a clear 'plaque' in the darkly stained tumour cell monolayer.

cThe 3T12 cells are easily distinguished from macrophages because they differ in shape, morphology, and by the presence of large nuclei with nucleoli.

d It is calculated that each well of 96-well plates contains 185 microscopic fields under these observation conditions. The average number of tumour cells in randomly selected fields is multiplied by 185 to calculate the total number of tumour cells per well

Was this article helpful?

0 0

Post a comment