Subsets of immune cells can be isolated before analysis by the enzyme-linked immunospot (ELISPOT) assay with various cell separation techniques. This chapter describes techniques to select desired cells or deplete unwanted cells by crosslinking cells to dense or magnetic particles for subsequent separation. The RosetteSep™ method can be used to isolate specific cell types directly from human whole blood, using the red blood cells (RBCs) present in the sample as dense particles. Unwanted cells are crosslinked to multiple RBCs, forming “rosettes.” The rosettes, free RBCs, and granulocytes pellet when the sample is centrifuged over a buoyant density medium. The unlabeled, desired cells are simply collected from the interface between the plasma and the buoyant density medium. The SpinSep™ method for isolation of mouse spleen or bone marrow cells is similar to RosetteSep, except that the unwanted cells are bound to dense particles rather than RBCs. The EasySep™ immunomagnetic system can be used with cell suspensions from a variety of species. Cells are crosslinked to nanometer-sized paramagnetic particles. Magnetically labeled cells are separated from unlabeled cells by placing the sample in a high gradient magnetic field. Both the labeled and the unlabeled fractions can be recovered for further use.