Cell Death Detection ELISAPLUS Protocol

Product No. CELLDETH-RO

Protocol

Use with non-proliferating cells

Since the Cell Death Detection ELISAPLUS assay does not require prelabeling of cells, it can detect internucleosomal degradation of genomic DNA during apoptosis even in cells that do not proliferate in vitro (for example, freshly isolated tumor cells). Use this kit for relative quantification of histone-complexed DNA fragments (mono- and oligonucleosomes) in the cytoplasm of cells after the induction of apoptosis (requires cell lysis) or released from necrotic cells into the cell culture supernatant or into serum/plasma.

Use of adherent cells

No change needs to be made for adherent cells. The lysis buffer is efficient enough for preparing the sample. The buffer can then be removed and put into microcentrifuge tubes and centrifuged, similar to the plate protocol.

Purpose of centrifugation step

Centrifugation at 200 x g is necessary before using the supernatant for detection of apoptosis in order to separate DNA fragments from other cell components (e.g., cell walls and cell nuclei which contain non-fragmented DNA). The non-fragmented DNA is not separated by centrifugation itself, but because the small DNA fragments (ladder) can escape by passing through the nuclei cell wall. Therefore, the supernatant contains the whole spectrum of the DNA ladder up to a maximum, which is too big to pass the nuclei wall. The length distribution is dependant on the cell type and also on the inducing agent.

This centrifugation step is only necessary for suspension cells. When working with adherent cells, simply remove the supernatant carefully and completely, and then lyse the cells directly into the microplate well.

Protein determination

The kit lysis buffer contains protein and detergents. Therefore, protein determination in the lysed samples is not possible.

Materials

     
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