Consider the following points carefully when planning your In-Cell Western™ Assay.
Proper selection of microplates can significantly affect results, because each plate has its own characteristics, including well depth, plate autofluorescence, and well-to-well signal crossover.
In-Cell Western Assays require sterile plates for tissue culture growth.
Before imaging, carefully remove any liquid from plate wells by aspiration.
The recommended focus offset for the recommended plates is 3.0 mm.
If you are using plates other than those recommended, the focus offset can be determined empirically by scanning a plate containing experimental samples and control samples.
Use the same intensity settings for each scan. After reviewing the scans, use the focus offset with the highest signal-to-noise for experiments. The actual minimum and maximum focus offset will vary with each instrument. Alternatively, consult the plate manufacturer to determine the distance to the bottom of the plate to help you begin the focus offset optimization process.
The Odyssey CLx AutoMode function alleviates the need to scan the plate at multiple intensity settings.
|Imager||Initial Intensity Setting (700/800 nm)||Intensity Settings Weak Signals||Initial Intensity Settings Saturated Signals|
|Odyssey Classic||5 / 5||7.5 / 7.5||2.5 / 2.5|
|Odyssey Sa||7 / 7||8 / 8||4 / 4|
|Aerius||7 / 7||8 / 8||4 / 4|
Protect plates from light before imaging to ensure highest sensitivity. When storing plates after imaging, the plates should remain protected from light at 4 °C.