Labeling cells with labeling antibodies and imaging with a fluorescent microscope is a staple technique in the Simon Lab. Our development of microfluidic flow chambers has allowed us to probe signaling pathways using fluorescent reporters in living neutrophils. In this technique, a neutrophil rolling on inflamed endothelial cells (or a substrate coated with similar adhesion molecules) is recorded in a rapid sequence of individual fluorescent images. For example, we have imaged the subcellular location of high affinity integrins in neutrophils migrating on endothelial cells, and found that the integrins redistribute on the cell surface following arrest, guiding the direction of migration.

Recently, we have begun to image neutrophils loaded with calcium sensitive dyes (i.e. Fluo-4). Using this method, we have determined that calcium influx is closely linked to the deceleration of rolling neutrophils to an arrest. Furthermore, we have found that the process of rolling on a selectin bearing surface increases the base-line calcium concentration. Simultaneous measurements of neutrophil rolling velocity and calcium concentration would clearly not be possible with a static imaging technique.

Videos (Quicktime)

CD18 Activation

TS2/4 Labeled migrating PMN:

LFA-1 localization and distribution on PMN crawling on recombinant ICAM-1 and E-selectin

Calcium Imaging in Real Time:

PMN labeled with Fluo-5f Calcium dye. Real time calcium imaging at contact sites of PMN arresting on ICAM-1 and E-selectin susbtrate by TIRF

Calcium Flux via IL8 stimulation:

fMLP triggered neutrophil arrest and calcium flux on endothelial cell monolayer

327C-Alexa 488

High affinity CD18 in unactivated neutrophils on recombinant ICAM-1 substrate