Phagocytosis Assay Kit (Zymosan Substrate) (ab211156)

Overview

  • Product name

    Phagocytosis Assay Kit (Zymosan Substrate)
    See all Phagocytosis kits
  • Detection method

    Colorimetric
  • Sample type

    Adherent cells
  • Assay type

    Cell-based
  • Species reactivity

    Reacts with: Mammals
  • Product overview

    Phagocytosis Assay Kit ab211156 uses a Zymosan substrate to provide a robust system for screening TLR ligands, phagocytosis activators or inhibitors. The phagocytosis assay protocol uses pre-labeled Zymosan particles as a pathogen for triggering phagocytosis.


    The engulfed Zymosan particles react with a specific substrate to produce a colorimetric signal that can be detected by absorbance at 405 nm. External particles are blocked before the reaction with a blocking reagent, ensuring the signal is directly proportional to the amount of internalized particles.


    This format provides a quantitative, high-throughput method to accurately measure phagocytosis, and avoids subjective manual counting of Zymosan particles inside cells.


    Phagocytosis assay protocol summary:
    - add Zymosan substrate to cell cultures and incubate for 15 min to 2 hrs
    - remove culture medium, and wash cells 
    - add fixation solution and incubate for 5 min and then wash cells
    - add blocking reagent and incubate for 60 min and then wash cells 
    - add permeabilization solution and incubate for 5 min and then wash cells
    - add detection reagent and incubate for 60 min and then wash cells
    - add detection buffer and incubate for 10 min
    - add substrate and incubate for 5-20 min
    - add stop solution and analyze with plate reader

  • Notes

    In mammals, phagocytosis by phagocytes (e.g., macrophages, dendritic cells, and neutrophils) is essential for a variety of biological events. Phagocytosis comprises a series of events, starting with the binding and recognition of particles by cell surface receptors, followed by the formation of actin-rich membrane extensions around the particle.  

    Zymosan (Saccharomyces cerevisiae) is prepared from yeast cell wall and consists of protein-carbohydrate complexes.  Zymosan is a commonly used pathogen in phagocytosis assays.

    The kit is suitable for adherent phagocytes only.

    Each 20 test kit provides sufficient quantities to perform 20, 10 or 5 tests in a 96-,48- or 24-well plate respectively. Each 96 tests kit provides sufficient quantities to perform 96, 48 or 24 tests in a 96-, 48- or 24-well plate respectively. Each 5 x 96 tests kit provides sufficient quantities to perform 5 x 96, 48 or 24 tests in a 96-, 48- or 24-well plate respectively.

  • Platform

    Microplate reader

Properties

  • Storage instructions

    Store at +4°C. Please refer to protocols.
  • Components 1 x 96 tests 5 x 96 tests 20 tests
    10X Permeabilization Solution 1 x 1.5ml 1 x 1.5ml 1 x 500µl
    250X Detection Reagent 1 x 50µl 1 x 50µl 1 x 20µl
    Blocking Reagent 1 x 200µl 1 x 200µl 1 x 50µl
    Detection Buffer 1 x 10ml 1 x 10ml 1 x 2ml
    Fixation Solution 1 x 20ml 1 x 20ml 1 x 2ml
    Phagocytosis Inhibitor 1 x 20µl 1 x 20µl 1 x 20µl
    Stop Solution 1 x 12ml 1 x 12ml 2 x 1.5ml
    Substrate 1 x 12ml 1 x 12ml 2 x 1.5ml
    Zymosan Suspension 1 x 1ml 1 x 1ml 1 x 250µl

Images

  • 50,000 cells/well of Raw 264.7 macrophages were seeded overnight in a 96-well plate.  Cytochalasin D was used to pretreat Raw 264.7 cells for 1 hr at 37ºC before addition of Zymosan particles at 50:1 ratio. Phagocytosis was stopped after 30 minutes and the amount of engulfed Zymosan particles was determined as described in the Assay Protocol.

Protocols

References

ab211156 has not yet been referenced specifically in any publications.

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