LUDOX-S40 must be used as a single point reference with the aim to attain a
ratiometric conversion factor, which in turn will be used to transform absorbance
data into standard OD 600 measurement.
Standard 1 cm path length spectrophotometer readings are instrument dependent,
while plate readers possess a path length less than 1 cm and are volume dependent.
Therefore, in this situation, there are 2 key objectives:
- Ratiometric conversion to transform Abs 600 measurements into OD 600
- Accounting for instrument differences
Before starting the protocol, path length correction must be switched off. This is
because scattering increases with longer wavelengths therefore adjustment is
confounded by scattering solutions such as dense cells. However, many plate
readers have automatic path length correction which is based on volume
adjustment that uses ratio of absorbance measurements at 900 + 950 nm.
LUDOX solution is only weakly scattering so will produce low absorbance values
Use same cuvettes, plates and volumes that are going to be used in cell based assays
- 1 mL 100% LUDOX
- H 2 O
- 96 well plate (black with flat, transparent/clear bottom)
- 100 µl of LUDOX should be added into wells A1, B1, C1 and D2 (or 1mL into
- 100 µl of H 2 O should be added into wells A2, B2, C2 and D2
- Measure absorbance 600nm of all samples in all standard measurement modes in instrument
- Record data