3.3.1. Sample preparation
The cells are seeded the day before analysis at 5–20 103 cells
in 100 ll per well in 96-well plates. After induction of cell death,
50 ll of cell supernatant is transferred to a new 96-well plate for
LDH assay, while the source 96-well plate containing the cells is
used for an MTT assay (see above) at a pre-defined time. Following
addition of 50 ll of reconstituted substrate mixture (CytoTox 96
Assay) and incubation for 30 min at room temperature, 50 ll of
stop solution is added to each well and absorbance is measured
at 492 nm. Results are expressed either as optical absorbance
values or converted to international units of enzyme with reference
to a standard curve (included as positive LDH control with
the CytoTox 96 assay).