Apo-ONE® Homogeneous Caspase-3/7 Assay

Protocol tips

Upstream tips	Protocol tips	Downstream tips
- Use identical cell numbers and volumes for the assay and the negative control samples. - Cells were cultured in normoxic and hypoxic conditions.		- The plates were incubated at room temperature and luminescence was measured at time 0, 30 and 60 min after the start of the assay,

Upstream tips
- Use identical cell numbers and volumes for the assay and the negative control samples. - Cells were cultured in normoxic and hypoxic conditions.
Downstream tips
- The plates were incubated at room temperature and luminescence was measured at time 0, 30 and 60 min after the start of the assay,

Publication protocol

The Apo-ONE® Homogeneous Caspase-3/7 Assay (Promega) was used to measure the activity of caspase-3 and −7, the key effectors of apoptosis. Cells were plated in 96-well plates as before with triplet repeats per plate (normoxic and hypoxic). The protocol was carried out according to the manufacturer’s instructions. Working in the dark, 10 µl of 100X substrate was added to 990 µl of buffer. Following hypoxic exposure, a 1:1 ratio using 40 µl of assay reagent was added to each well containing 40 µl of sample medium. The plates were incubated at room temperature and luminescence was measured at time 0, 30 and 60 min after the start of the assay, using a Promega GloMax® plate reader (Promega) at 499 nm (excitation) and 521 nm (emission).

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Manufacturer protocol

Download the product protocol from Promega for Apo-ONE® Homogeneous Caspase-3/7 Assay below.

Download manufacturer protocol

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