Development of a novel flow cytometric cell-mediated cytotoxicity assay using the fluorophores PKH-26 and TO-PRO-3 iodide

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Abstract

A flow cytometric (FCM) assay has been developed for the determination of cell-mediated cytotoxicity (CMC). In the assay, the target tumour cell population was labelled with a membrane dye, PKH-26, prior to incubation with splenocyte effector cells. Cell death within the target population was assessed by the addition of the viability probe TO-PRO-3 iodide (TP3) and analysed by flow cytometry. The extent of cytotoxicity was determined by the relative number of live target cells labelled with PKH-26 only and dead, permeabilised cells labelled with both PKH-26 and TP3. This CMC method allows the analysis to be conducted on a single cell basis and overcomes the need for radiochemicals. This communication indicates that the FCM assay is an accurate and reproducible experimental system capable of analysing natural killer (NK) cell and antibody-dependent cell-mediated cytotoxicity. The procedure is comparable to the chromium release assay. We believe that this is one of the first demonstrations of an FCM-based antibody-dependent cell-mediated cytotoxicity (ADCC) assay.

Introduction

The immune system is able to recognise and destroy target cells, such as tumours and virally infected cells, through the mechanism of cell-mediated cytotoxicity (CMC). A distinct set of lymphocytes known as natural killer (NK) cells play a prominent role in this process. These cells do not possess antigen-specific receptors, but are capable of killing via two distinct pathways. Firstly, NK cells possess receptors, which enable them to recognise and, subsequently, lyse cells that either lack or have down-regulated MHC class I molecules. Alternatively, the expression of the FcγIII receptor (CD16) allows NK cells to recognise IgG bound to the antigen on the target cell surface. This process is known as antibody-dependent cell-mediated cytotoxicity (ADCC) (Perussia, 1998).

The most commonly used method for measuring CMC is the chromium-51 (51Cr) release assay. This involves radioisotope-labelling of the target cells with 51Cr, which is passively taken up and binds to intracellular proteins. Cell lysis is measured by the release of the radioactive probe into the culture supernatant (Brunner et al., 1968). Although this method is widely used, it has disadvantages in that 51Cr is expensive and volatile, there may be high spontaneous release of the probe, certain cell types are difficult to load and, finally, radioisotopes may have a detrimental effect upon cell function.

Flow cytometric (FCM) assays have been developed in an attempt to overcome some of these difficulties and confer the advantage of allowing the analysis to be conducted on a single cell basis. Initial attempts exploited differences in light scatter properties to identify targets from effector cells (Vitale et al., 1989). However, in several cases, problems with discrimination between the two cell populations were encountered (Ellis, 1993). Radosevic et al. (1990) determined NK cell cytotoxicity by labelling target cell membranes with the green fluorescent dye, F-18 in combination with the DNA intercalating dye, propidium iodide (PI), to evaluate target cell death. Recently, the red fluorescent membrane dye, PKH-26, has been used in preference to F-18, as it labels with a higher degree of reliability and is stable over longer periods of time in vitro Hatam et al., 1994, Lowdell et al., 1997. There is a high degree of overlap in the emission spectra of PKH-26 with PI and, although compensation may be applied, this overlap can be problematic, especially with strongly labelled cells.

In this paper, we describe a modified FCM assay using PKH-26 to label target cells in combination with the viability probe TO-PRO-3 iodide (TP3) Van Hooijdonk et al., 1994, O'Brien and Bolton, 1995. Similar to PI, TP3 binds nuclear DNA, but has an advantage over PI in that its peak absorbance is 642 nm with a peak emission of 661 nm and is best excited by a red diode laser emitting at 633 nm (Hirons et al., 1994). As there is no overlap between the emission spectra of TP3 and PKH-26, compensation is not required. Four cell populations may be clearly distinguished, namely, live targets, dead targets, live effectors and dead effectors. Furthermore, this method offers the potential for the measurement of up to two additional fluorochromes, such as FITC and PE/Cy5.

Section snippets

Antibodies

Antibodies utilised in ADCC assays were the hamster monoclonal anti-murine MHC class II antibody, N22 (ATCC no. HB-225) and polyclonal hamster IgG (cat. no. 11111D, Becton Dickinson, UK) as a control antibody.

Target cell preparation

The murine target cell lines chosen were the T-cell lymphoma, RMA (C57BL/6, H-2b) (Graham et al., 1995), the B-cell lymphoblast, BCL1-3B3 (BALB/c, H-2d) (ATCC no. CRL-1669) (Gronowicz et al., 1980) and the NK cell sensitive lymphoma, YAC1 (A/Sn, H-2a) (ATCC no. TIB-160) (Sjogren and

Identification of target cells using fluorescent probes

The lipophilic cell membrane dye PKH-26 offers a simple method by which candidate tumour cell lines may be labelled, enabling them to be differentiated from unlabelled cells by FCM (Fig. 1A). Cell death can be determined by uptake of the DNA intercalating dye TP3. In order to investigate the potential of TP3 as a viability probe, cells were treated with the permeabilising agent, PermeaFix™ and probed with either TP3 or PI. Subsequent analysis showed not only that the fluorophore TP3 could be

Discussion

An in vitro CMC assay has been developed in which target and effector populations can be visualised and differentiated while measuring cytotoxicity within target cells. Previous analysis of NK cell function and ADCC has relied mainly upon radioisotope-based CMC. This study describes a novel FCM-based method for assessing in vitro cell killing using the membrane dye PKH-26 and the DNA intercalating viable dye, TP3. In the assay, PKH-26 is used to label and distinguish targets from effectors and

Acknowledgements

The financial support of Antisoma is gratefully acknowledged. We are also grateful to Dr. David Flavell (Southampton General Hospital, Southampton) for donating YAC1 cells and Dr. Mark W. Lowdell (Royal Free Hospital, London) for technical advice with assay design.

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