Optimization of conditions for specific binding of antibody-coated beads to cells.
It has previously been demonstrated that cells can bind antibody-coated beads; this effect can be used to enhance the fractionation of cells using magnetic fields or by centrifugation on isopycnic, isotonic density gradients. As a general rule, the higher the expression of surface antigens the more beads bind to cells. However, we have also noted that other factors also affect the number of beads found bound to cells. Experiments have been carried out to determine what factors affect binding of antibody-coated beads to cells. The optimum conditions for binding of antibody-coated beads to MOLT-4 T cells were found to be, namely, a 20:1 bead to cell ratio in a 1 ml incubation volume, with continuous end-over-end mixing for 1 h at 25 degrees C. Furthermore, the optimum centrifugation conditions at which the samples were separated on isopycnic, isotonic density gradients were determined as 220 x gmax for 90 min, at 20 degrees C. The results indicate the preferred conditions that are necessary to achieve optimum bead binding by cells and their subsequent fractionation. Different antibody-coated beads were examined including Dynabeads M-450, used as a known standard. In addition we describe, as a possible alternative to Dynabeads, dense polystyrene beads, for the separation of cells on the basis of the immunological identity of the surface of cells using density perturbation methods.