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Location-dependent artifact for no measurement using multiwell plates

J C Snell1, C A Colton, O N Chernyshev

  • 1Department of Physiology and Biophysics, Georgetown University Medical School, Washington, DC, USA.

Free Radical Biology & Medicine
|January 1, 1996
PubMed
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The Griess reaction for measuring nitric oxide (NO) production is affected by a "border effect" in multiwell plates. Regulating the gaseous environment eliminates this artifact, improving NO measurement accuracy.

Area of Science:

  • Biochemistry
  • Cell Biology
  • Analytical Chemistry

Background:

  • The Griess reaction quantifies cellular nitric oxide (NO) production by measuring nitrite levels in cell supernatants.
  • Standard methods involve subtracting background nitrite from a blank, which is usually adequate.
  • However, this approach is unreliable for multiwell plates, especially with low NO production or long incubations.

Purpose of the Study:

  • To investigate the cause of variable nitrite levels observed in cell-free wells of multiwell plates during NO production assays.
  • To identify and mitigate the
  • border effect
  • affecting NO measurements in 96-well plates.

Main Methods:

  • Utilized the Griess reaction to measure nitrite levels in cell-free media within a 96-well plate.

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  • Compared nitrite accumulation in edge wells versus interior wells.
  • Manipulated the gaseous environment by altering air exchange.

    • Main Results:

    • Significant and variable nitrite accumulation was detected in cell-free media from the edge wells of 96-well plates.
    • Media from interior wells showed no detectable nitrite accumulation.
    • Controlling gaseous exchange, specifically reducing air exchange, eliminated the observed "border effect".

    Conclusions:

    • The "border effect" in NO measurements using the Griess reaction in multiwell plates is attributed to non-cellular nitrite formation.
    • Nitric oxides (NOx) in ambient air are the likely source of this artifact.
    • Regulating the gaseous environment is crucial for accurate NO quantification in such assays, particularly for edge wells.