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An All-on-chip Method for Rapid Neutrophil Chemotaxis Analysis Directly from a Drop of Blood
Published on: June 23, 2017
Development of a new method of analysing chemotactic deactivation of human neutrophil granulocytes
1Department of Dermatology, University of Kiel, Schittenhelmstr. 7, D-24105, Kiel, Germany. obock@dermatology.uni-kiel.de
Abstract:
Measurement of chemotactic migration of human neutrophil granulocytes (PMN) induced by chemotaxins serves as a simple and reliable method for assessing the expression of chemotaxin receptors. Incubation of PMN with a certain chemotaxin leads to a diminished chemotactic migration towards this chemotaxin. This is called chemotactic deactivation. We developed a new deactivation chamber to determine chemotaxis and chemotactic deactivation of human PMN. This novel chamber is a modification of the commercially available acrylic 48-well microchemotaxis chamber consisting of an upper block with wells drilled all the way through the block and a blind-well lower block. Both blocks are separated by a polycarbonate membrane. PMN from the wells in the upper block migrate through the pores of the membrane into the wells of the lower block containing the chemoattractants. Migrated PMN on the lower side of the PC membrane were quantified after staining by measuring specific light absorbance. The chemotactic activity is quantified as a ratio of stimulated migration and random migration (chemotactic index=CI). For our novel chamber, only the upper blocks of this commercial chamber were connected like a sandwich, including a polyvinylpyrrolidone-free polycarbonate membrane with a pore size of 3 microm. The wells in the upper compartment were filled with 5 x 10(4) PMN and deactivating chemotaxin. The lower block was then filled with the chemotactic stimulus and the chamber was then incubated in humidified air with 5% CO2 atmosphere at 37 degrees C. The influence of cell concentration, incubation time, chemotactic factor concentration, pore size and alkaline treatment of polycarbonate membranes on migrational activity of PMN have been investigated. The technique was rigorously standardized in order to optimize the assay conditions. The method is relatively simple, sensitive and fast. The determination of chemotaxis and deactivation are performed in the same chamber, thus avoiding cell loss due to nonspecific adherence in other incubation tubes. The chamber can be used to characterize the chemotactic activity of chemoattractants of unknown structure via known and unknown receptors. This new chamber can be very helpful in detecting unknown chemotactic stimuli, which are not detectable by, for example, antibodies.
Insights
Researchers developed a novel deactivation chamber for precise measurement of human neutrophil granulocyte (PMN) chemotaxis and deactivation. This method offers a simple, sensitive, and fast way to assess chemotactic receptor function and identify unknown chemotactic stimuli.
Area of Science:
- Immunology and Cell Biology
- Biomedical Engineering
- Assay Development
Background:
- Chemotactic migration of human neutrophil granulocytes (PMN) is crucial for immune response and assessing chemotaxin receptor expression.
- Chemotactic deactivation, a diminished migration response after prior exposure to a chemotaxin, is a key phenomenon.
- Existing methods for measuring chemotaxis and deactivation can be complex and prone to cell loss.
Purpose of the Study:
- To develop and validate a novel deactivation chamber for simultaneous measurement of PMN chemotaxis and chemotactic deactivation.
- To optimize assay conditions for sensitivity, speed, and reliability in quantifying PMN migration.
- To enable characterization of chemoattractants and detection of novel chemotactic stimuli.
Main Methods:
- A modified 48-well microchemotaxis chamber was designed with an upper and lower block separated by a polycarbonate membrane.
- Human PMN were incubated with a deactivating chemotaxin in the upper wells, and chemoattractants were placed in the lower wells.
- Migrated PMN were quantified via light absorbance after staining, with chemotactic activity expressed as a chemotactic index (CI).
Main Results:
- The novel chamber allows for direct determination of both chemotaxis and chemotactic deactivation in a single assay.
- Influences of cell concentration, incubation time, chemotactic factor concentration, pore size, and membrane treatment were investigated and standardized.
- The method is demonstrated to be simple, sensitive, fast, and minimizes non-specific cell adherence.
Conclusions:
- The developed deactivation chamber provides a robust and efficient platform for studying PMN chemotaxis and deactivation.
- This technique facilitates the characterization of known and potentially unknown chemotactic stimuli and their receptors.
- The chamber offers a valuable tool for immunological research and drug discovery by enabling the detection of novel chemotactic factors.
