Granulocyte chemotaxis: multiple assay screening using a raft technique.

Abstract

SUMMARY The assessment of granulocyte chemotaxis is complicated by the difficulty of precisely reproducing results in serial estimations and deciding on the best end point which would reflect most accurately the degree of travel taken by the cells under observation. The methods in use are generally based on the Boyden chamber;' following this, we have further developed the principle of the "raft" technique of chamber based migration.2 In order to overcome the problems associated with reproducibility of results when performing multiple assays of chemotaxis, especially when sera of widely differing activity are encountered in the screening procedure, we have used a "batching" system and a simple method of presenting the results so that they are comparable.The means of quantifying the degree of granulocyte migration most commonly employed is the "leading front" method;3 we have found this unsatisfactory even at 90 min for faster-moving granulocytes or stronger chemoattractants, as some of the cells move beyond the membrane and may be lost to assay.34 In these circumstances, three possible solutions exist:(i) the micropore membrane could be thickermany authors5 6 say they have been unable to find membranes of standardised thickness and quality and until manufacturers can correct this and provide thicker membranes this solution remains theoretical.(ii) use a weaker chemotaxin; sometimes assays are done for the express purpose of looking for decreased migration-for example, when an inhibitor is present; in such cases designated chemotaxins need to be used against the special nature of the inhibitor-for example, C5a for C5a specific inhibitor.7 8 Thus the second solution has limited application.(iii) a shortening of incubation time from 3 h to around 60 to 90 min has already been employed by all the investigators using the "leading front" estimation.However, if one is looking for inhibitors, then the chemotaxin should give relatively long leading fronts so that shortening can be more clearly observed. In our work we regularly use an attractant which produced a leading front of at least 100,um in 90 min.Some of the difficulties were overcome by Maderazo and Woronick5 who described a leucotactic index (LI) which required that the incubation Accepted for publication 17 June 1981 time be flexible between 60 and 90 min to prevent the leading front from penetrating the whole of the permeable membrane (see later). We describe a method of assessment using a uniform incubation time which appears to retain the sensitivity of the LI but requires no time adjustments and a simple apparatus. It is best suited for multiple assays especially where there is a wide range of chemotactic activity in the sera to be examined-especially on a serum prior to further evaluation depending on the results obtained. This method of recording and presenting results also illustrates the pattern of migration. Material and methodsNormal human group 0 granulocytes from dextran sedimentation of whole blood are washed twice in Hanks basal salt solution ...