Using CytoTox-ONE kit

Cytotoxicity assay

Overview

Cells are plated, incubated with phenol (or lysis solution) at different concentrations and then LDH levels are measured to determine the relative levels of cell death/damage at each concentration. Include 3 replicates of each phenol concentration with cells and 3 with no cells as controls.

Each reaction contains (for 96 well plates):

Plated cells (or no cells for controls)
100uL Phenol Stock (150mM) + Media in different ratios (or Lysis solution + Media)
100uL CytoTox-ONE reagent

Process

Remove media from plated cells.
Add 100uL of appropriate Phenol dilution or Lysis dilution to each well (table below).
Incubate 2 hours.
Add 100uL CytoTox-ONE to each well.
Immediately read wells with excitation at 560nm and emission at 590m. Record at least 3 absorbance measurements at least 1 minute apart.

Phenol Stock : Media ratios

% Phenol stock (mM Phenol)	150mM Phenol (uL)	Media (uL)
50% (75mM)	50	50
40% (60mM)	40	60
30% (45mM)	30	70
20% (30mM)	20	80
10% (15mM)	10	90
0% (0mM)	0	100

Note: Phenol LD50 (at 2 hours) for isolated rat hepatocytes is 12mM (Moridani et al. 2003)

Lysis Solution : Media ratios

Lysis Solution (uL)	Media (uL)
2	98

Viability assay

Overview

Cells are plated and LDH levels are measured after different amounts of time (~12 hours apart) to determine the relative levels of cell death/damage over time. Include 3-4 replicates for each time point.

Each reaction contains (for 96 well plates):

Plated cells (or no cells for controls)
100uL Media in different ratios
100uL CytoTox-ONE reagent

Process

Change media in plated cells, adding 100uL media to each well.
Add 100uL media to no-cell control wells.
At each timepoint:
- 100uL CytoTox-ONE reagent to each well for that timepoint.
- Immediately read wells with excitation at 560nm and emission at 590m. Record at least 3 absorbance measurements at least 1 minute apart.

RECIPES

Phenol Stock (150mM)

For ~10mL:

158uL Phenol (89%)
10mL Media (the same media the cells are in)