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Cell-based Flow Cytometry Assay to Measure Cytotoxic Activity
Published on: December 17, 2013
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A flow cytometry based assay that simultaneously measures cytotoxicity and monocyte mediated antibody dependent
Aljawharah Alrubayyi1, Alexandra Schuetz2, Kerri G Lal1
1U.S. Military HIV Research Program, Walter Reed Army Institute of Research, Silver Spring, MD, USA; Henry M. Jackson Foundation for the Advancement of Military Medicine, Inc., Bethesda, MD, USA.
Journal of Immunological Methods
|August 28, 2018
Summary
The rapid and fluorometric ADCC assay measures antibody-dependent cellular cytotoxicity (ADCC) but is confounded by monocyte phagocytosis. A new combined assay distinguishes monocyte and NK cell antibody effector functions.
Area of Science:
- Immunology
- Cellular immunology
- Antibody effector functions
Background:
- Antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis (ADCP) are critical immune responses.
- The RV144 trial highlighted the importance of non-neutralizing antibodies in HIV prevention.
- The mechanism of the rapid and fluorometric ADCC (RFADCC) assay remains unclear.
Purpose of the Study:
- To investigate the mechanism of the RFADCC assay.
- To differentiate between monocyte-mediated phagocytosis and NK cell-mediated cytotoxicity in antibody effector functions.
- To develop a combined assay for simultaneous measurement of these functions.
Main Methods:
- Utilized the RFADCC assay with PKH26 dye.
- Combined RFADCC with amine-reactive dye staining for target cell killing.
- Correlated RFADCC and amine-reactive assay results with established NK cell assays.
Main Results:
- Monocytes acquire PKH26 dye in the RFADCC assay, indicating phagocytosis.
- RFADCC readout correlates with phagocytosis, not solely cytotoxicity.
- The combined assay successfully distinguished between monocyte and NK cell antibody effector functions.
- Amine-reactive assay results correlated with NK cell-specific antibody effector functions.
Conclusions:
- The RFADCC assay readout is influenced by monocyte phagocytosis.
- A novel combined assay allows simultaneous discrimination of monocyte and NK cell antibody effector functions.
- This assay provides a more accurate assessment of specific antibody-mediated immune responses.
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