Phototoxicity and fluorotoxicity combine to alter the behavior of neutrophils in fluorescence microscopy based flow adhesion assays

The use of fluorescent probes that allow visualization of leukocyte-endothelial cell (EC) interactions has greatly informed our understanding of leukocyte recruitment. However, effects of these agents on the biological functions of leukocytes are poorly described, leading to concerns about the interpretation of such data. Here we used two flow-based neutrophil adhesion assays to compare the effects of phase contrast illumination (PCI) with high intensity illumination (HII) used for fluorescent microscopy, in the presence or absence of five commonly used fluorochromes. Isolated neutrophils were either (1) perfused across P-selectin to establish a population of rolling cells, which were subsequently activated with fMLP; or (2) perfused across EC activated with TNF-alpha. In the absence of fluorescent dyes, HII did not affect levels of leukocyte adhesion; however, subsequent neutrophil behavior was dramatically altered when compared with cells under PCI, for example, dramatically reducing their migration velocities. In the presence of fluorescent dyes, the effects of HII were exacerbated, although the precise nature of the biological effects of these probes was agent specific. Thus, for the first time, our experiments describe the effects of fluorescent microscopy on the separate stages of the neutrophil recruitment process and reveal a previously unsuspected effect of HII on neutrophil migration.