Perhaps the most simple, cheap, and effective technique for in vitro cytotoxicity tests is the one based on target ceil labelling with Na251Cr04. This is probably still the most widely used method and is commonly referred to as 'the chromium (MCr) release assay'. Radioactive chromate passes through the cell membrane by an unknown mechanism and is bound mainly to cyto-
plasmic proteins. Since 51Cr is a y-emitter, no special preparation of cell samples is needed for radioactive determination. This technique was first used to evaluate the effect of radiation on thymocytes (43) and complement-mediated cytotoxicity (44), and was subsequently applied to the study of cellmediated cytotoxicity (45). This method measures lysis of target cells, so both apoptotic and non-apoptotic deaths are detected.
Protocol 1. Chromium-51 release assay Safety recommendations
Chromium-51 is a medium-energy -y-radiation emitter and it must be manipulated according to the corresponding national regulations for use of radioactive materials. The researcher must wear appropriate protective clothing (laboratory coat, surgical gloves) and dosimeter. Stock, concentrated solutions of Na251Cr04 must be conveniently shielded (half-value layer: 3 mm lead, approximately). On the other hand, the manipulation of
51Cr does not require any other special precaution, since in the form of chromate it is not absorbed by any organ in the body.
Equipment and reagents
• tissue culture facilities • V-shaped 96-well microtitre plates
• effector and target cells • sterile Na251Cr04 in aqueous solution (from
• complete culture medium (e.g. RPMI 1640 Amersham, NEN, or ICN) (1 mCi/ml) supplemented with heat-inactivated 5% • -y-radiation counter (solid scintillation) FCS, 5 mM Hepes, pH 7.4, and antibiotics)
The protocols given are adequate for labelling of tumour target cells and for lymphocytes from mixed lymphocyte cultures (MLC). Some examples of commonly used murine target cells, including their MHC restriction, are the following:
• L1210: lymphocytic leukaemia-expressing H-2d
• EL-4: thymoma-expressing H-2Kb
• RMA: lymphoma-expressing H-2Kb
• P815: monocytic leukaemia-expressing H-2d
• L929: fibrosarcoma-expressing H-2Kk (adherent cell) Two labelling alternatives are possible.
1. Resuspend the appropriate amount of washed, cultured targets in non-diluted FCS at 1 x 106 cells in 100 ^l.
2. Add 10 n.Ci of Na251Cr04 per 1 x 10® cells and incubate at 37°C for 1 h.
3. Wash then by dilution and centrifugation in complete medium, resuspend at 1 x 10® cells/ml in complete medium, and incubate for another hour at 37 °C.
4. Eliminate the second supernatant, wash again in complete medium, and resuspend the cells for the experiment.
(b) Overnight labelling
1. Resuspend around 2 x 10® target cells in 5 ml of complete medium, place the suspension in a 25 cm2 culture flask, and add 200-300 |xCi of Na251Cr04.
2. Incubate overnight at 37°C in a C02 incubator, and then wash twice with RPMI 1640.
3. Resuspend the cells in complete medium for the experiment.
1. The manipulation during the cytotoxicity tests can be done under nonsterile conditions. Resuspend the labelled target cells at 105 cells/ml and add 100 |xl per well of 96-well plates, so that 104 labelled target cells are used per experimental point. Make points at least in triplicate.
2. Wash and resuspend the effector cells in complete medium. Several effector to target (E:T) ratios should be used. In the case of CTL clones, E:T ratios that should give a good lysis are between 1:1 and 10:1. In the case of primary mixed lymphocyte cultures (MLC), the E:T ratios recommended are between 20:1 and 100:1. Resuspend the effector cells at the maximum cell density to be used in a given experimental point. Then, plate 100 jjlI on the wells already containing the labelled target cells, previously having made the corresponding dilutions of the effectors with complete medium to obtain the E:T ratios chosen.
3. Include at least triplicates of (i) labelled target cells alone resuspended in 200 |jil of complete medium; and (ii) labelled target cells alone in 100 jxl of complete medium where 100 jjuI of 1% Triton X-100 or 2 N HCI are added to estimate, respectively, the spontaneous release and total labelling of the targets.
4. Centrifuge the plate at 400 g at room temperature for 1 min to favour cell-cell contact. Incubate at 37°C in a cell Incubator for the time chosen. Usually, 4 h cytotoxicity tests are performed, but longer times can also be used, provided that spontaneous release of the label from the targets does not increase above 20% of the total labelling. If spontaneous release in the experimental conditions used is greater than 30%, the assay is not reliable.
5. At the end of the incubations, centrifuge the plates at 400 g during 5 min and harvest 100 (J aliquots of each supernatant by using a multichannel pipette. Transfer the aliquots directly to small RT-15 plastic tubes (Bibby Sterilin), seal them with molten paraffin wax, and determine the radioactivity associated with the supernatants in the 7-counter.
6. Calculate the specific 51Cr release at the different E:T ratios as follows: % of lysis = % of specific 51Cr release =
(cpm sample-cpm spontaneous release) ^ (cpm maximum release-cpm spontaneous release)
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