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Cell death detection in Xenopus embryos by ELISA Cell death detection in Xenopus embryos by ELISA

Veenstra et al. (1998) Cell Death Diff. 5:774-784
This protocol uses the Cell death detection ELISA reagents from Roche / Boehringer Mannheim. Submitted by Gert Veenstra.

Sample preparation

Wash embryos 1 x in 25% MMR

Remove excess buffer

Add 10 volumes "incubation buffer", i.e. 50 µl for 5 embryos

Lyse the embryos by gently pipetting up and down using a large blue tip
        Use a negative control, as shearing of genomic DNA may give false positive signals

Dilute 10 µl lysate with 190 µl "incubation buffer"
        Consider testing other dilutions

Incubate this sample for 30 min. at 4°C

Centrifuge at 13,000 rpm (eppendorf) for 10 min. at 4°C

Remove 160 µl supernatant / embryo, avoiding pellet contamination of the supernatant

Samples can be stored, if not used the same day, in aliquots at -20°C

Do ELISA according to manufacturer's instructions.