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Plate Selection and Instrument Settings

Proper selection of microplates can significantly affect results, as each plate has its own characteristics, including well depth, plate autofluorescence, and well-to-well signal crossover. Use the general considerations for microplate selection provided below.

  • In-Cell Western analyses use detection at the well surface with no liquid present. This results in minimal well-to-well signal spread, allowing the use of both clear as well as black-sided plates with clear bottoms. Do not use plates with white walls, since autofluorescence from the white surface will create significant noise.
  • In-Cell Western assays require sterile plates for tissue culture growth. The following plates are recommended by LI-COR® Biosciences:
    • 96-well format, Eppendorf (P/Ns 0030730119 or 0030741030)
    • 96-well, clear, Nunc® (P/Ns 167008, 161093)
    • 96-well, clear, Falcon (P/Ns 353075, 353948)
    • 96-well, black with clear bottom, Nunc (P/N 165305)
    • 96-well format, Corning (P/N 3603)
    • 384-well, clear, Nunc (P/Ns 164688, 164730)
    • 384-well, clear, Falcon (P/Ns 353229, 353289)
    • 384-well, black with clear bottom, Nunc (P/N 142761)
    • 384-well format, Corning (P/N 3712)
  • Focus Offset Optimization - The focus offset can be determined empirically by scanning a plate containing experimental and control samples using the following focus offset settings, if plates other than those recommended above are used.
Instrument Resolution Focus Offset Scan Quality Intensity Setting
(700/800 nm)
Scan Time Medium Quality
Odyssey® Classic 169 μm 3.5 medium-lowest 5/5 7 min
Odyssey CLx 169 μm 3.5 medium-lowest 5/5 7 min
169 μm 3.5 medium-lowest AutoScan 16 min
Odyssey Sa 200 μm 3.5 medium-lowest 7/7 3 min
Odyssey Aerius 200 μm 3.5 medium-lowest 7/7 3 min

Use the same intensity settings for each scan. After reviewing the scans, use the focus offset with the highest signal-to-noise ratio for experiments. The actual minimum and maximum focus offset will vary with each instrument. Alternatively, the plate manufacturer can be consulted to recommend the measured distance from the skirt to the bottom of the plate.

  • All Aerius and Odyssey® Imaging systems (excluding Odyssey Fc) require microplates that have a maximum 4.0 mm distance from the base of the microplate to the target detection area of the plate (actual maximum focus offset varies with each Aerius and Odyssey Sa instrument and is found by choosing Settings > System Administration, then clicking Scanner Information). When using plates specified for In-Cell Western assays, the recommended focus offset is 3.5 mm or higher.

Intensity Setting Optimization

Instrument Intensity Setting
(700/800 nm)
Intensity Settings: Weak Signal(700/800 nm) Intensity Settings: Saturated Signal(700/800 nm)
Odyssey Classic 5/5 7.5/7.5 2.5/2.5
Odyssey CLx 5/5 7.5/7.5 2.5/2.5
Odyssey Sa 7/7 8/8 4/4
Odyssey Aerius 7/7 8/8 4/4

*The Odyssey CLx AutoScan function alleviates the need to scan the plate at multiple intensity settings.

  • 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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