The following methods are proven to work by PEVIVA AB and should be used as guidelines when collecting samples.

A. SEEDING OF CELLS

Day 1: Seed the cells. The seeding density has to be worked out for each cell type and agent to be used. For example in a human breast cancer cell line, 10⁴ cells were seeded per well in a 96-well plate. In general, the test has sufficient sensitivity to detect the M30 neo-epitope from less than 1,000 apoptotic cells.

Day 2: Wash the cells once with PBS and add fresh medium. Expose the cells to pro-apoptotic treatment as desired.

Note: When cells are seeded the day before the start of an experiment, fresh medium should be added the next day since M30-reactivity may accumulate after seeding the cells due to spontaneous apoptosis.

B. Culture supernatants

Day 1: Seed in cells according to section A: Seeding of cells.

Day 2-4: Collect medium samples (2x25 µl are used for each assay). When collecting multiple samples from the same culture, add fresh medium back to the culture.

If the assay will be performed within 24 hours, the samples should be stored at 2-8°C. Samples to be analyzed later should be stored below -20°C. Avoid repeated freeze-thawing.

Before analysis by ELISA, centrifuge the samples at 4°C at 5,000 rpm for 2 minutes in a tabletop centrifuge. Collect the supernatant.

M30-reactivity has been demonstrated in tissue culture supernatants 8 hours after addition of pro-apoptotic agents like staurosporine. However, after the addition of genotoxic agents, longer incubation times (>24 hours) were required to obtain M30-reactivity in culture supernatants.

C. Cell extracts

Day 1: Seed in cells according to section A: Seeding of cells.

Day 2-4: Examine the cells under the inverted microscope. If the cells are attached, remove the medium. Add ice-cold lysis buffer (10 mM Tris-HCl, pH 7.4/10 mM MgCl₂/150 mM NaCl/0.5% NP-40).

Use 100 µl - 1.5 ml, depending on the number of cells and the type of culture vessel. First add 50% of the desired volume to the cell monolayer, allow lysis (on ice) for 5 minutes and transfer the extract to a centrifuge tube. Then wash the dish/multiwell plate with the remaining 50% of the lysis buffer. Add this volume to the previously collected extract and centrifuge at 4°C at 5,000 rpm for 2 minutes in a tabletop centrifuge. Transfer the cytosol extract (supernatant) to a new tube and store at -20 to -70°C. Samples to be assayed within 3 months can be kept at -20°C, otherwise samples should be stored at -70°C.

If the cells have detached, collect the medium and pellet the cells by centrifugation. Remove the supernatant and add the lysis buffer. Vortex and incubate on ice for 5 minutes. Centrifuge at 4°C at 5,000 rpm for 2 minutes and freeze the cytosol as described above. Avoid repeated freeze-thawing of extracts.

M30-reactivity has been demonstrated in cell extracts a few hours after addition of pro-apoptotic agents. For many agents, 16-24 hours are a suitable timing. In cell extracts, very high M30-reactivity can be obtained (beyond the range of the standard curve). It may therefore be necessary to dilute such samples before assay using the provided Diluent (Vial H).

D. Total activity in cell extracts and culture supernatants

For some applications such as drug screening, it may be advantageous to screen for total M30-reactivity at one, late time point. It is possible to assay total activity in media and extracts by adding non-ionic detergent directly to the media.

Day 1: Seed in cells according to section A: Seeding of cells.

Day 2-4: For 96-well plates containing 200 µl medium per well: add 10 µl 10% NP-40 per well. Allow lysis to occur on a rotatory shaker for 5 minutes at room temperature. Use 25 µl for M30-Apoptosense® test.

Optional: transfer the extracts to V-bottom 96 well plates and clear by centrifugation at 4°C at 5,000 rpm for 2 minutes in a centrifuge. Use 25 µl for M30-Apoptosense® test.

Note:

- Fresh medium should be added when collecting multiple samples from the same culture.

- Do not use contaminated samples.

- If tissue culture media are stored at -20°C for prolonged periods, they may become alkaline. This can be avoided by adding 0.1 volume of 1 M Tris-HCl, pH 7.4 before freezing.

E. Serum and plasma specimens

Enough blood should be collected before, during and after exposure to be sufficient for 2x25 µl serum (duplicates) at each assay. Donors need not to be fasting prior to blood collection and no special preparations are necessary.

Collect blood by venipuncture, avoiding hemolysis, into plain tubes (without anti-coagulant) and separate the serum from the cells. M30-Apoptosense® can also be used for plasma samples. M30-values are comparable for serum and plasma samples when appropriate dilution is accounted for.

If the assay will be performed within 48 hours, the sample should be refrigerated at 2-8°C. If testing will be delayed more than 48 hours, the sample should be divided into aliquots and stored frozen below -20°C.

Frozen samples should be thawed only once for 1 hour at 20±4°C and sera thoroughly mixed after thawing. Avoid repeated freeze-thawing. Do not use samples that are contaminated.