Assay of Chemotaxis by a Reversible Boyden Chamber Eliminating Cell Detachment

Abstract

A modified in vitro assay for measuring chemotaxis of leucocytes is presented. A closed chamber is used in preference to an open one thus allowing reversal of the chamber during the incubation. The responding cells which at the time the chamber is turned over are inside the filter, then migrate upwards and are consequently retained on the filter by gravity. This chamber is compared with the conventional type and various parameters are examined using guinea pig eosinophils and other cell types. Loss of cells in the conventional Boyden technique is negligible for rabbit peritoneal neutrophils but is considerable for human and guinea pig neutrophils and eosinophils. This is circumvented by the proposed method; accordingly it allows more accurate quantitation in clinical and experimental work.

Monocyte and Neutrophil Chemotactic Activity of Normal and Diluted Human Serum and Plasma

“…Chemotaxis Assay A modified Boyden chamber described in a previous paper [9] was adapted for use of human cells. It consists of two closed compartments which allows the chamber to be turned upside down during the experiment in order to permit re sponding cells to migrate upwards and thus pre vent cell detachment after migration.…”

“…It consists of two closed compartments which allows the chamber to be turned upside down during the experiment in order to permit re sponding cells to migrate upwards and thus pre vent cell detachment after migration. Filter pro cessing and other details of methodology were as described [9].…”

Different Concentrations of Chemotactic Factors Can Produce Attraction or Migration Inhibition of Leukocytes

“…Chemotaxis was measured using the reversible chemotaxis chamber recently described [13]. This chamber overcomes cell detachment from the fil ter after migration, thus eliminating the possibility that a 'fast' response provoked by a strong stimu lus is not detected because the cells have already detached from the filter when the incubation is terminated.…”

“…Such a reversing has been shown to be an appropriate maneuver. However, since dislodg ing of rabbit neutrophils has earlier been shown to be minimal [13], chambers needed not to be re versed in rabbit neutrophil experiments. Unless otherwise stated, incubation times, pH values, fil ter processing and other details of methodology were as described previously [13].…”

“…Such a cellular effect could be expressed as inability of the cells to rec ognize the stimulus if presented in too high a concentration or in a response of the rec ognizing cells other than unidirectional mi gration such as migration inhibition. There fore, chemotactic agents have been tested over a broad concentration range in parallel in the Boyden chamber assay [6,13], and the classical migration inhibition capillary tube test [8,11]. Casein as both a monocyte and neutrophil cytotaxin and acid-dena tured purified anaphylatoxin [28] attracting neutrophils but not macrophages served as chemotactic agents.…”