51-Chromium Labeled Polymorphonuclear Leukocyte Chemotaxis

A New Modification of Boyden Chamber Method

Abstract

Neutrophil chemotaxis is an important mechanism in the first stage of the inflammatory response against the invasion of microorganism in human and other species. During the past 100 years many investigations have looked at the fundamentals of neutrophil chemotaxis in vitro and went on to develop many types of assay to quantify neutrophil chemotaxis in the laboratory. In 1962, Boyden reported a new method of neutrophil chemotaxis with a special chamber with a lower compartment, where a chemoattractant was placed separated by a filter on top of which a cell suspension was placed.
In searching for a sensitive and more conv inient method of neutrophil chemotaxis, we have modified the previous method of 51-chromium labeled neutrophil chemotaxis.
Normal human subjects, aged 24 to 37 years, donates the blood. Polymorphonuclear cells were purified by using Ficoll-Hypaque and dextran sedimentation. Final chromium uptake was determined to be 2.78±0.98 % of the original count. We used acrylic blind well chemotactic chambers which were divided using 3 or 5, um pore sized polycarbonate filter (Nuclepore) (13mm diameter) on top of 3 or 5 μm pore sized cellulose ester filter (Millipore)(13mm diameter) and compared to original method. To find the optimum incubation time for chemotaxis using a 5 μm Nuclepore on top of the 5 μm Millipore, we counted all of the compartments of this chamber system. By varying incubation time from 15 to 150 minutes, we noted that the neutrophils begin migration within 15 minutes. At 60 minutes, a marked difference was detected in the lower filters of the chambes with EAP vs media. As incubation time was lengthened mere counts were found in the lower filters and less counts were found in the upper chamber fluids. Chemotactic activity was correlated with the cell concentration from 2.875×10⁵ cells/ml to 6.9×10⁶ cells/ml. In our 22 experiments using 5, μm Nuclepore filters with 5 pm Millipore filters, the target: non target ratio was 4.68±1.83when incubated for 90min.
These results sho w that our method using Nuclepore on top of a Millipore filter reduced incubation time from 3 hours to 90 minutes while maintaining successful chemotaxis. A shorter incubation time maximizes laboratory efficiency by reducing spontaneous 51-chromium release from injured and dying cells yielding more reproducible results and also by allowing a laboratory to test more samples in less time.