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Experimental Design Considerations

Consider the following points carefully when planning your In-Cell Western Assay.

Plate Selection

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.

Plate Color

  • Use black-sided plates with clear well bottoms. In-Cell Western analyses use detection at the well surface with no liquid present. This results in minimal well-to-well signal spread.
  • Do not use plates with white walls, because autofluorescence from the white surface will create significant noise

Sterile Plates

In-Cell Western Assays require sterile plates for tissue culture growth.

Image Settings

Before imaging, carefully remove any liquid from plate wells by aspiration.

Focus Offset

The recommended focus offset for the recommended plates is 3.0 mm.

Determine Focus Offset

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.

Intensity Setting Optimization

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 CLx AutoMode - -
Odyssey Sa 7 / 7 8 / 8 4 / 4
Aerius 7 / 7 8 / 8 4 / 4

Plate Storage

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.

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