Supplemental Material: Blot Rolling Demonstration video
- Supplemental Data for "Direct Real-Time Observation of E- and P-Selectin Mediated Rolling on Cutaneous Lymphocyte-Associated Antigen Immobilized on Western Blots" by
Robert C. Fuhlbrigge, Sandra L. King, Charles J. Dimitroff, Thomas S. Kupper, and Robert Sackstein
Blot Rolling Demonstration Video
This video shows P-selectin-mediated rolling adhesions on human CLA immobilized on a Western blot. To prepare the blot, lysate of human CLA-positive T cells was subjected to SDS-PAGE, blotted onto PVDF membrane, and stained with HECA-452 mAb as described in Materials and Methods. The blot was incorporated into the parallel plate flow 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 as shown in Fig. 1. The apparatus was mounted on the microscope stage with the field of view oriented over the upstream (rightmost or higher molecular weight) portion of the HECA-452-stained band at ~140 kDa (see Fig. 2) (stain is not apparent under the conditions used for this recording). CHO-P cells were introduced into the chamber under continuous shear flow and allowed to form tethers for 1 min at 0.53 dyne/cm2 (0.14 ml/min). The flow is then increased to 1.75 dyne/cm2 (0.46 ml/min) and maintained at this rate for the remainder of the observation period. For brevity, the video sequence provided begins after the shear rate has been increased. CHO-P 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 (CLA) band. As cells reach the downstream edge they can be observed to detach from the blot and continue downstream at hydrodynamic velocity. No rolling cells are observed in the blot area below the stained band.
