Collection And Preparation Of In Vitro Samples For Research Use Only - peviva M30 Apoptosense ELISA Gebrauchsanweisung

Instructions for Use
Store samples at 2 – 8 °C up to 4 hours. For longer periods, store samples frozen
at -20 °C or lower. Samples can be freeze-thawed without loss of activity (ref. 6,
7), but it is recommended that repeated freeze-thawing should be avoided. For
dilution of samples see sections "Component Preparation" and "Performance
Characteristics".
Collection and Preparation of in vitro Samples
for Research Use Only
M30 Apoptosense ELISA has been used for cell-culture applications in a number
of published studies (see www.vlvbio.com for references). Peviva has developed
a specific product for in vitro cell cultures, M30 CytoDeath™ ELISA (PEVIVA Prod.
No. 10900). This product has a dynamic range and sensitivity suitable for in vitro
work.
The following protocols can be used for detection of apoptosis of cultured epi-
thelially derived cells using the M30 Apoptosense ELISA.
Sample preparation from cell cultures
For many applications, it is advantageous to measure total M30 reactivity (ccK18)
at a single, late time point. Such measurements reflect an integrated assessment
of apoptosis. To assay total ccK18 fragments in cell culture media and cell extracts,
add non-ionic detergent directly to the cells in the tissue culture medium.
Day 1: Seed the cells. The seeding density needs to be determined for the spe-
cific cell type and the type of cytotoxic agent; 5 000–10 000 cells per well in a
96-well plate is usually adequate.
Day 2: Wash the cells once with PBS and add fresh medium (200 µL/well). Expose
the cells to the desired agent(s).
Day 2 – 4: For 96-well plates containing 200 µL of medium per well, add 10 µL
of 10 % NP 40 per well. Allow lysis to occur on a rotatory shaker for 5 minutes
at room temperature. Mix gently by pipetting up and down, careful not to cre-
ate air bubbles, and transfer 2 × 25 µL of the medium/lysate to the wells of M5
Coated Microstrips.
Sample preparation from cell culture supernatants
The M30 Apoptosense ELISA and M65® ELISA can be used to assess cell death
mode by calculation of an M30:M65 ratio (ref. 3). Such measurements should
be performed using medium supernatants! The ratio should be calibrated for
each carcinoma cell line using appropriate controls, i.e. agents known to induce
apoptosis (e.g. genotoxic agents, staurosporine) and/or mainly necrosis (e.g. oli-
gomycin/glucose starvation or hydrogen peroxide).
Day 1/Day 2: Seed the cells, wash and add agents as described above.
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