Preparation of effector cells

1 Resuspend monocytes/macrophages at 2 x 10s cells/ml in complete medium.

• Combitips (Eppendorf)

• Complete medium (see Protocol J)

• Ca2+-, Mg21 -free Dulbecco's modified PBS (see Protocol 1)

• 0.0556 (w/v) trypsin, 0.02% EDTA solution (Hyclone Laboratories)

• Macrophage effector cells, appropriately purified (see Chapters 1 and 2)

• Tumour target cells: e.g. the P815 murine mastocytoma cell line, maintained in complete medium by serial passages on 100 mm tissue culture dish or in 75 cm2 tissue culture flasks

• Macrophage activators (see Protocol 1)

Protocol 2 continued

2 Pipette 0.1 ml aliquots of the cell suspension (containing 2 x 10s cells) into triplicate wells of 96-well U-bottom tissue culture plates using a repeating dispenser.

3 Add macrophage activators (as described in Protocol i).

4 After 18-24 h incubation, wash cells extensively (as described in Protocol i).

B slCr labelling of target cells

1 Wash the P815 murine mastocytoma tumour target cells once with complete medium and resuspend them at a concentration of5 x 106/ml in complete medium.

2 Label with 5lCr by incubating 5 x 106 cells in 1 ml of medium with 500 |iCi ofs,Cr in a 50 m! conical tube for 1 h at 37 °C with occasional shaking/

3 Wash radiolabeled tumour cells twice with 50 ml complete medium to remove non-incorporated label.

4 Return labelled ceils to the incubator for 1 h at 37°C to allow the release of non-incorporated label into the medium. This 'cold chase' decreases spontaneous release of label during the assay.

5 Wash the cell suspension once more just before addition to macrophage cultures.

6 Adjust concentration to 2.5 x 104 cells/ml in complete medium.

C Cytotoxicity assay

1 Add 0.2 ml of target cell suspension (containing 5 x 103 51Cr-labelled cells) to wells containing washed monocyte/macrophage monolayers to give a 40:1 E:T cell ratio. Labelled tumour cells should also be added to wells without macrophages to determine the spontaneous releaseb and the total incorporated counts/

2 Incubate the test plates in a humidified atmosphere containing 5% CO, at 37 °C for 18 h.

3 At the end of the incubation time, centrifuge the plates at 300 g for 5 min.

4 Collect 0.1 ml of supernatant/well with an automatic pipette and assess the radioactivity of each sample in a gamma counter.

5 Express the results of the cytotoxicity assay as the percentage of specific 51Cr release, calculated from the average of triplicate samples, according to the following formula: { (experimental c.p.m. - spontaneous c.p.m.) (total c.p.m. incorporated in target cells - spontaneous c.p.m.) }x 100.

Experimental c.p.m. is the radioactivity released in wells containing macrophages and target cells; spontaneous c.p.m. is the radioactivity released by target cells cultured alone; total release is the radioactivity from lysed target cells.

J Any kind of leukaemia, lymphoma, or mastocytoma cell, either in suspension or adherent, can be used as tumour target cells in this assay.

bThe spontaneous release in the 18h5ICr release assay is typically 30-35% of total radioactivity, cTotal incorporated counts are estimated by adding 20 ^I/well of 10% SDS or 50 p. 1/we 11 of 0.1 M NaOH or 1% (v/v) Triton X-100 to lyse cells. The average counts of three wells containing the target cells is used.

There are a few variations of the 31Cr release assay for macrophage-mediated cytotoxicity, A faster assay with a lower spontaneous release employs TNFa-sensitive target cells, such as the WEHI 164 murine fibrosarcoma or the MOPC-315 murine plasmasytoma cell lines (see Section 3) (9,12-14). In this procedure tumour cells at the concentration of 1 x lOs/ml are pre-treated with 1 ^g/ml of actinomycin D for 3 h at 37 °C in a C02 incubator, washed twice with complete medium, and then radiolabeled as described above. 51Cr-labelled cell concentration is adjusted so that 5 x 103 cells in 0.1 ml are added to each well of the 96-well plate containing 2 x 105 effector cells in 0.1 ml. The release of radiolabel from tumour target cells is determined after a six hour incubation period.

2.2.2 [3H]TdR and [125l]dUrd release assays

Nuclear labels, such as [3H]thymidine (TdR) and [1jiododeoxyuridine (dUrd), have a low spontaneous release (less than 10-15% of the total incorporated counts over 72 hours) and have been widely used in assays of 48 hours or longer (15-18). Macrophage cytotoxicity assays with [:iHJTdR and [125I]dUrd pre-labelled tumour cells require actively dividing target cells for incorporation of the label into DNA, and the level of radioactivity may vary in different cells. In addition, the release of nuclear labels requires both cell death and autolysis which results in delayed detection of cell killing and requires assays of longer duration to quantitatively reflect cytolysis. Because [3H]thymidine (TdR) is reutilized to some extent after release by target cells and/or it is taken up by macrophages, the percentage of lysis determined does not yield values that reflect the real extent of tumour cell killing and the artificially low release may give misleading results (17-19). [nsI]dUrd, on the other hand, is not reutilized but can inhibit target cell proliferation and is toxic for certain target cells (4, 15,16).

Protocol 3 describes a cytotoxicity assay that can be used with either [3H|TdR- or [,25I]dUrd-labelled target cells

Protocol 3

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