[125IIododeoxyuridine 125IUdR release assay

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125IUdR competes with thymidine for incorporation during DNA synthesis because the iodine atom is sterically similar to the methyl group of thymidine. So 125IUdR is incorporated into DNA and, when the target cell is killed by effector cells, 125IUdR is released in the form of DNA fragments that are not re-utilized. Since this event is a nuclear marker of apoptosis, 125IUdR release is the method of choice to analyse nuclear apoptosis during cell-mediated cytotoxicity. This method was initially used by comparing it with 51Cr release to distinguish CTL-induced death from lysis induced by antibody plus complement, which is not associated with DNA fragmentation (46,47).

Protocol 2. [125l]lododeoxyuridine (125IUdR) release assay Precautions

125l is a low-energy -y- emitter, and is more easily shielded (half-value layer: 0.02 mm lead) than 51Cr. The same precautions as indicated for 51Cr manipulation should be used here (Protocol 1). The incorporation of 125l into the thyroid by inhalation is its main biological risk. However, since

125IUdR is not volatile, special precautions are not needed for its manipulation.

Equipment and reagents

• tissue culture facilities

• effector and target cells

• sterile 1.5 ml Eppendorf cones

• culture medium (e.g. RPMI 1640 supplemented with heat-inactivated 5% FCS, 5 mM Hepes, pH 7.4, and antibiotics)

A. Labelling of target cells

1. It is convenient for target tumour cells to be in an exponential phase of growth when labelling with 125IUdR.

• sterile ,26IUdR (from Amersham, NEN, or ICN) (1 mCi/ml)

• hypotonic lysis buffer: 0.5% Triton X-100 in 20 mM Tris, 1 mM EDTA, pH 7.5

• 7-counter (solid scintillation counter)

2. Resuspend the washed target cells in complete medium at 5 x 105 cells/ml.

3. Add 10-15 pxCi of 125IUdR per ml and incubate in a C02 incubator at 37°C for 2-3 h. Then, wash the cells twice with RPMI 1640 and resuspend in complete medium for the experiment.

B. Cytotoxicity test

1. Resuspend the labelled target cells at 1 x 105 cells/ml and add 100 jjlI to a 1.5 ml Eppendorf tube, so that 10* labelled target cells are used per experimental point. Each experimental point should be made in triplicate at least.

2. Wash and resuspend the effector cells in complete medium. The same considerations about E:T ratios indicated for the 51Cr release assay (Protocol 1) apply here. Resuspend the effector cells at the maximum cell density to be used in a given experimental point. Then, add 100 |jd into the tubes already containing the labelled target cells, having previously made the corresponding dilutions of the effectors with complete medium to obtain the E:T ratios chosen.

3. Include duplicates, at least, of labelled target cells alone, resuspended in 200 jjlI of complete medium to estimate the spontaneous release of the label from the targets.

4. Centrifuge the Eppendorf tubes at 400 g at room temperature for 2 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 is not greater than 20% of the total labelling.

5. Centrifuge the tubes at 400 g for 5 min, harvest carefully the whole supernatant, and save it for counting.

6. Lyse the cells by adding 500 (d of ice-cold lysis buffer, followed by brief vortexing, and incubation on ice for 20 min. Centrifuge the lysates at 12000 g for 10 min at 4°C in a minifuge, collect the super-natants containing fragmented DNA and cut off (with scissors or blade) the bottom of the tube that contains non-fragmented DNA.

7. Determine the radioactivity associated with each sample in a y-counter, including the incubation medium, the supernatant of the lysate, and the DNA pellet.

8. Calculate the specific 125IUdR release at the different E:T ratios as follows:

% DNA fragmentation = % of specific 125IUdR release =

(cpm sample-cpm spontaneous)

X 100

(cpm total-cpm spontaneous)

cpm sample = (cpm in the incubation medium + cpm in the cell lysate) of the sample cpm spontaneous = (cpm in the incubation medium + cpm in the cell lysate) spontaneous cpm total = (cpm in the incubation medium + cpm in the cell lysate + cpm in the DNA pellet) of the sample

2.3 The JAM test

This method measures, using target cells labelled with [3H]thymidine and a conventional cell-harvester, the intact DNA that remains associated with living target cells. This allows for the estimation of the DNA amount that has undergone fragmentation during cell-mediated cytotoxicity (48). Hence, the JAM test detects DNA fragmentation induced by effector cells on the labelled targets and the data obtained can be considered equivalent to those obtained using the 125IUdR release assay.

Protocol 3. [3H]Thymidine-labelled DNA retention by living cells

Precautions

3H radioactivity is a low-energy fj-radiation emitter. Conventional plastic and glass containers and surgical gloves protect quite well from this type of radiation. On the other hand, due to its low energy, tritium p-particles must be detected in a liquid scintillation counter, making the sample preparation slightly more laborious. This method is fast, sensitive, and easy to perform if an automated cell-harvester is available, but it generates more radioactive waste.

Equipment and reagents

• tissue culture facilities

• effector and target cells

• sterile round-bottom, 96-well plates

• culture medium (e.g. RPMI 1640 supplemented with heat-inactivated 5% FCS, 5 mM Hepes, pH 7A, and antibiotics)

• sterile [3Hlmethylthymidine (from Amer-sham, NEN, or ICN) (1 mCi/ml)

• automated cell-harvester

• scintillation vials and liquid scintillation fluid

• p-counter (liquid scintillation counter)

A Labelling of target cells

1. It is very convenient if the target cells to be in an exponential phase of growth when labelling with [3H]thymidine.

2. The day before the assay, resuspend the target cells into fresh medium and add 1 ml to the wells of a 24-well tissue culture plate at 3-5 x 105 cells/ml.

3. To label, add [3H]thymidine to a final concentration of 5 |xCi/ml to the cultures and incubate under the same culture conditions for 4-6 h. After the incubation, wash the cells twice with RPMI 1640 and resuspend in complete medium for the experiment.

B Cytotoxicity test

1. Resuspend labelled target cells at 105 cells/ml and add 100 |xl per well, so that 104 labelled target cells are used per experimental point. Each experimental point should be made in triplicate at least.

2. Wash and resuspend the effector cells in complete medium. The same considerations about E:T ratios indicated for the previously described assays also apply here. Resuspend the effector cells so that 100 |xl contains the maximum effector number to be used in a given experimental point. Add 100 |jlI of the corresponding dilution of effector cells to the labelled target cells, in order to obtain the desired E:T ratios.

3. Include at least triplicates of labelled target cells alone, resuspended in 200 pj of complete medium to estimate the spontaneous release of label from the targets. To calculate the total labelling, a triplicate of labelled target cells can be directly placed into scintillation vials.

4. Centrifuge the plates at 400 g at room temperature for 2 min to favour cell-cell contact. Incubate at 37°C in a C02 incubator for the time chosen (1-4 h).

5. At the end of the incubations, aspirate the cells and medium from the plate wells on to fibre glass filters using an automated cell-harvester.

6. Wash the filters, dry, and count them in a liquid scintillation counter.

7. Calculate the percentage of DNA fragmentation at the different E.T ratios as follows:

% specific DNA fragmentation =

(spontaneous cpm-sample cpm) ^ spontaneous cpm spontaneous cpm = cpm from retained labelled DNA in the absence of killers sample cpm = cpm from retained labelled DNA in the presence of killers

Although the total incorporation (total labelling of target cells in the absence of effector cells) is not needed for the calculation of specific DNA loss, it does need to be measured in each assay, to be sure that the DNA retained in the absence of killers ('spontaneous cpm') is roughly the same as the total labelling.

2.4 BLT-esterase release assay

One of the consequences of CTL activation through the antigen receptor or by the combination of phorbol esters and calcium ionophores is the exocytosis of cytoplasmic granules containing perform and granzymes. The presence of these proteins in the supernatant of stimulated CTL can be detected by a colorimetric method termed the BLT-esterase release assay (49). Trypsin-type serine esterases are detected in fact in these assay, granzyme A being predominant in CTL or NK cells.

Protocol 4. BLT-esterase release assay

Equipment and reagents

• tissue culture facilities

• effector and target cells

• culture medium (e.g. RPMI 1640 supplemented with heat-inactivated 5% FCS, 5 mM Hepes, pH 7.4, and antibiotics)

• flat and round-bottomed 96-well plates

• ELISA plate reader

Assay

1. Wash and resuspend the effector cells in complete medium. Stimulate the cells with the appropriate stimulus. The stimuli could vary: anti-TCR/CD3 antibodies, phorbol esters plus calcium ionophores, alloantigen-bearing target cells, etc. If target cells are chosen as stimulants, several effector to target (E:T) ratios may be used. In this case, the E:T ratios recommended are 1:1, 1:2, and 1:3, increasing the number of targets, not of effectors, to optimize granule release from the CTL. In this case, resuspend effector cells in complete medium at 2 x 10® cells/ml and add 50 n,l of the suspension (1 x 105 cells) into the wells of a round-bottomed 96-well plate. Resuspend the target cells so that 50 |xl contains the maximum target cell number to be used in a given experimental point and then make the corresponding dilutions with complete medium. Plate 50 |xl of the target cell suspensions on the wells that already contain the CTL, in order to obtain the E:T ratios chosen.

2. Include at least triplicates of (i) effector cells alone resuspended in 100 |xl of complete medium; and (ii) effector cells alone in 50 n,l of complete medium where 50 |xl of 1% Triton X-100 are added to estimate, respectively, the spontaneous release and total CTL content of BLT-esterase.

3. Centrifuge the plate at 400 g at room temperature for 2 min to favour cell-cell contact. Incubate at 37°C in a cell incubator during 3-4 h.

• benzyloxycarbonyl-L-lysine thiobenzyl ester (BLT) (from Sigma or Calbiochem) and dithiobis-(2-nltrobenzoic) acid (DTNB) (from Sigma). BLT powder should be stored at -20°C inside a desiccator, while DTNB can be stored at room temperature.

4. At the end of the incubations, centrifuge the plate at 400 g for 10 min, harvest carefully 30 of the supernatants, and plate them on the wells of a clean, flat-bottomed, 96-well plate. Make a triplicate blank with 30 julI of fresh culture medium.

5. During the last centrifugation, prepare the BLT reagent by dissolving 4 mg of BLT and 4 mg of DTNB in 50 ml of phosphate-buffered saline, pH 7.4, by gently shaking and vortexing. Add 150 (j,l of the reagent to each well of the plate and incubate at 37 °C. The BLT reagent is of limited stability and can only be used for 3 or 4 h after preparation.

6. Read the intensity of the yellow colour formed using an ELISA plate reader at 405-415 nm, depending on the filters available, subtracting automatically the blank values from the sample values. It is recommended that absorbances be read after different incubation times, for example a first reading at 15 min, followed by measurements at 30 min, 45 min, and 1 h.

7. Calculate the specific esterase release at the different E:T ratios as follows:

% of specific BLT-esterase release =

[(A««) sample-(^\405) spontaneous] X 100 [(/4405) total-(/A405) spontaneous]

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