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Related Experiment Videos

A cell-detachment solution can reduce background staining in the ELISPOT assay.

Angela Grant1, Sarah Palzer, Chris Hartnett

  • 1R&D Systems Inc., Minneapolis, MN, USA.

Methods in Molecular Biology (Clifton, N.J.)
|June 7, 2005
PubMed
Summary

Accumax effectively removes cells in many Enzyme-linked immunospot (ELISPOT) assays, improving cell removal without impacting results for several cytokine assays. However, it is incompatible with certain interferon-gamma ELISPOT assays.

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Comparing Flow Cytometry and ELISpot for Detection of IL-10, IL-6, and TNF Alpha on Human PBMCs.

Methods in molecular biology (Clifton, N.J.)·2024

Area of Science:

  • Immunology
  • Cell Biology
  • Biotechnology

Background:

  • Enzyme-linked immunospot (ELISPOT) assays detect cytokine-releasing cells by visualizing spots on porous membranes.
  • Cell adherence to membranes complicates washing and can lead to background noise or inaccurate spot counting.
  • Efficient cell removal is crucial for optimizing ELISPOT assay performance and data interpretation.

Purpose of the Study:

  • To evaluate the efficacy of Accumax, a cell detachment reagent, for removing cells adhered to microplate membranes in various ELISPOT assays.
  • To determine the compatibility of Accumax across a range of human, mouse, rat, and canine cytokine ELISPOT assays.
  • To assess whether Accumax treatment hinders or improves ELISPOT assay performance.

Main Methods:

  • Accumax was tested in 16 different ELISPOT assays, including those for human IL-2, IL-4, IL-5, IL-6, IL-8, IL-13, IL-1beta, IFN-gamma, and TNF-alpha.

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  • Assays were also performed using mouse IL-4, IL-6, IFN-gamma, TNF-alpha; rat IL-2, IFN-gamma; and canine IFN-gamma.
  • The impact of Accumax on cell removal, staining intensity, and spot formation was analyzed.

    • Main Results:

    • Accumax successfully removed a significant number of adhered cells in human IL-13, IL-1beta, IL-2, IL-4, IL-5, IL-8 and mouse IL-4, IL-6, TNF-alpha ELISPOT assays.
    • The reagent maintained assay performance in these compatible assays.
    • Accumax was incompatible with human, mouse, rat, and canine IFN-gamma ELISPOT assays, reducing staining intensity and spot counts.

    Conclusions:

    • Accumax is a valuable tool for improving cell removal in specific ELISPOT assays, particularly for certain interleukins and TNF-alpha.
    • The reagent's utility is limited in IFN-gamma ELISPOT assays due to detrimental effects on staining and spot detection.
    • Careful consideration of the specific cytokine and species is necessary when using Accumax in ELISPOT assays.