Czerlinski G H, Reid D S
Northwestern University, Chicago, IL 60611.
J Biochem Biophys Methods. 1987 Nov;15(2):85-95. doi: 10.1016/0165-022x(87)90036-4.
Monoclonal antibody 10.2-16 is directed toward the mouse class II major histocompatibility complex gene product 1-Ak expressed on the cell line LK35.2. Instead of activating cells by fluorophor we used (acrylamide-coated) heavy and magnetic microspheres of 0.6 micron in radius. These microspheres are chemically coupled (carbodiimide method) with the antibody toward the surface antigen. The cells are observed through a microscope with horizontal alignment, as they sediment in a (temperature controlled) tube with square cross-section. Stokes Law allows the determination of the density of cells (first alone) using viscosity and density of Dulbecco's modified Eagle's Medium together with the observed mean sedimentation velocity (66 microns/min) and a mean diameter of 10 microns. We found a density of 1.0558 +/- 0.0028 g/cm3 at 10 degrees C. Independently, thinly coated, heavy (and magnetizable) microspheres with the cited antibody are attached to cells and observed likewise. The increased sedimentation velocity permits us to show that the cells were fully covered with microspheres (290 per cell). A magnetic field gradient opposing gravity moved these cells against gravity with two different mean velocities, 340 microns/min and 850 microns/min. The higher velocity resulted in 290 particles per cell, the lower one in 130 particles per cell. The limits for the expansion of this method to smaller particle sizes (down to 10 nm) are evaluated.
