|Blot Rolling Demo
This video shows E-selectin-mediated rolling adhesions on HCELL (Hematopoietic Cell E-and L-selectin Ligand; sialofucosylated glycoform of CD44) from human hematopoietic KG1a cells immobilized on a Western blot. To prepare the blot, immunoprecipitated CD44 from membrane preparations of KG1a cells was subjected to SDS-PAGE, blotted onto PVDF membrane, and stained with HECA-452 mAb. The blot was incorporated into the parallel plate flow chamber apparatus using media with 10% glycerol with the gel origin and inlet port to the right and the dye front and outlet port to the left. Chinese hamster ovary cells permanently transfected to express E-selectin (CHO-E cells) were introduced into the chamber under physiological shear stress conditions (1 dyne/cm2). For brevity, the video sequence provided shows footage after tethering and rolling has been initiated. CHO-E cells are observed entering from the right and traversing the field of view at hydrodynamic velocity. Attached cells can be observed to roll at a much-reduced rate in the direction of flow. After several seconds observation, the field of view is moved downstream in overlapping steps covering the entire region of the HECA-452-stained 100kDa CD44 band (stain is not apparent under the conditions used for this recording). As cells reach the trailing edge of HECA-452 stain, they detach from the blot and continue downstream at hydrodynamic velocity.
This is a representative movie of T cell tethering, rolling, locomotion and, finally, transendothelial migration (TEM) on HUVEC under shear conditions in the presence of apical CXCL12 (10 ng/ml) and basal CCL5 (100 ng/ml). 8.5 minutes of a 10 minute trial are shown, compressed to 14.17 seconds. This field shows many T cells undergoing adhesive interactions with HUVEC and completing TEM. The green circle indicates a particular T cell performing TEM (the dominant cell group in this arrangement). Note that the indicated cell arrests on the endothelium and completes TEM (note change in T cell contrast to pale gray with passage through endothelium) within 5 minutes of time.