Cytotoxicity assay

1 Add 1 x 104 pre-labelled target cells in 0,2 ml of complete medium into each well of a 96-well plate containing 2 X 105 effector cells (E:T cell ratio of 20:1). Labelled tumour cells should also be added to wells without macrophages to determine the spontaneous release (release by tumour cells alone) and the total incorporated counts,*5

2 After 48-72 h in culture, harvest 0,1 ml of supernatant from each well with an automatic pipette.

3 When |3H]TdR is used, place supernatants in vials containing 5 ml of scintillation fluid and count released radioactivity in a beta counter; when [,Z5I]dUrd is employed, assess directly the amount of radioactivity of each sample in a gamma counter.

4 Calculate lytic activity as described in Protocol 2.

3 Almost every adherent mouse fibrosarcoma or adenocarcinoma can also be used as tumour target cells in this assay.

bTarget cells should have no more than 10000 c.p.m. per 4 x 10" cells, |3H]TdR or [125l]dUrd incorporation above this level may be toxic.

cThis 'cold chase' decreases the label in cytoplasmic pools and, in turn, decreases spontaneous release of label during assay.

d Spontaneous release is usually 10-20% of total incorporated radioactivity. Total incorporated counts are estimated by adding 20 ^1/well of 10% SDS or 50 ^il/well of 0.1 M NaOH to lyse cells. The average counts of three wells containing the target cells are used.

2.2.3 mlndium release assay mIndium (1111n | is an isotope widely used in nuclear medicine, recirculation, and lymphocyte-mediated cytolysis studies. It is a gamma emitter that labels most cell types efficiently with no decrease in cell viability, and localizes in the cytoplasm with up to 80% of the incorporated label quickly released upon target cell destruction. Spontaneous release is veiy slow (0.25-0.5% per hour) and this isotope can be used for both short- and long-term cytotoxicity assays (20-24). "'In release assay can be used to quantify macro ph age-media led cytolysis of a variety of target cells, both adherent and non-adherent, that generally exhibit similar labelling and spontaneous release profiles and whose destruction well correlates with levels of 11'In release. Rccausc of its high labelling efficiency and low toxicity, as low as 2-10 c.p.m. can be used for cell labelling, allowing assays employing as few as 10! target cells. The 24 hour11 'In release assay can substitute J1Cr in every application and has the advantage of a much lower spontaneous release that allows the extension of the cytolytic assay to 72 hours. The possibility to perform short- and long-term cytotoxicity assays using the same isotope offers a good system to examine the kinetics and mechanism of macrophage tumour-icidal activity at different levels of activation. For these reasons, we routinely use the l11ln release assay for testing both human monocyte and murine macrophage tumouricidal activity (20, 21, 23)

For use in cell labelling, 11'In must be com pi ex ed to 8-hy droxy qui noli ne (oxine) to form the mIn-oxine chelate (u'lnOx). This chelate is highly lipophilic and labels cells by rapid, passive diffusion. Even though the half-life of111 InOx is only 2.8 days, each commercial preparation can be used over a two week period because of the high activity of the isotope. In order to standardize the labelling technique, a known amount of ll1InOx is employed to label target cells. This is done by calculating the concentration of the isotope at the time of labelling from a calibration table and then by adding the appropriate volume containing the desired number of microcuries of the isotope. The cell lines of choice for this procedure are the P815 murine mastocytoma cell line for murine macrophage-mediated cytolysis, and the HT29 human colon carcinoma cell line for human monocyte-mediated killing. The recommended labelling time should be strictly observed because longer incubation can lower the viability of some cell lines. Moreover, only cell suspension of good viability should be labelled with 1 nlnOx because considerable amounts of the label, which is passively lipophilic, is incorporated by dead cells, therefore elevating baseline controls during the assay.

Protocol 4 describes an efficient method for monitoring macrophage-mediated tumour cell lysis using mindium release.

Protocol 4

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