FIGURE 14.1 Cell Separation by Density. (a) Cells are layered on to a preformed gradient (see Fig. 14.2) and the tube centrifuged. The tube is placed on a gradient harvester, and flotation medium (e.g., Fluorochemical FC43) is pumped down the inlet tube to the bottom of the gradient, displacing the gradient and cells upward and out through the delivery tube into a multiwell plate. The cells are diluted with medium (so that they will sink) and cultured. (b) Purified HeLa cells recovered from an artificial mixture of HeLa and MRC-5 finbroblasts. (c) Purified MRC-5 eluting at a lower density. (Gradient harvester after an original design by G. D. Birnie.)

FIGURE 14.2 Gradient Former. Two chambers are cut in a solid transparent plastic block and connected by a thin canal across the bases of the chambers and exiting to the exterior. A delivery tube with a stainless steel tip, long enough to reach the bottom of the centrifuge tube, is inserted in the outlet. With the valves closed, the left-hand chamber is filled with high-density medium and the right-hand chamber with low-density medium. Valve 1 is opened, the stainless steel tip is placed in the bottom of the tube, the stirrer is started, and then valve 2 is opened. As the solution in the left chamber runs into the right, it mixes and gradually increases the density, while the mixture is running out into the tube.

FIGURE 14.3 Centrifuge-Derived Gradient. Gradient generated by spinning Percoll at 20,000 g for 1 h.

FIGURE 14.4 Centrifugal Elutriator Rotor (Beckman Coulter). A cell suspension and carrier liquid enter at the center of the rotor and are pumped to the periphery and then into the outer end of theseparating chamber. The return loop is via the opposite side of the rotor, to maintain balance.

FIGURE 14.5 Magnetic Sorting. Negative sort. Committed progenitor cells from bone marrow suspension bound to Dynal paramagnetic beads with antibodies to lineage markers. Lineage-negative (stem) cells are not bound and remain in the suspension ready for sorting by flow cytometry. (a) Inserting the tube into the magnetic holder. (b) Tube immediately after being placed in magnetic holder. (c) Tube 30 s after placement in magnetic holder. (See also Plate 23a).

FIGURE 14.6 Magnetic Cell Sorting (MACS Technology). Positive sort. (a) Cells are preincubatedwith antibodies raised against a cell-type-specific surface antigen and conjugated to paramagneticMACS MicroBeads. (b) When the cells are introduced into the column, cells bound to MicroBeads are retained in the magnetic field generated by magnet and column matrix; unlabeled cells go straight through. (c) The magnetically bound cells, released when the column is removed from the magnet,are flushed out with the piston. (See also Plate 23 d, e). (d) Dot plot from flow cytometry of tumor cellculture containing fibroblasts before MACS sorting (top), flow-through fraction after MACS sorting with Anti-Fibroblast MicroBeads, as in (b), enriched fibroblast fraction (bottom) after releasing from magnet. (See also Fig. 14.7b, c.) (d, © Miltenyi Biotec, used with permission.)

®

FIGURE 14.7 Fluorescence-Activated Cell Sorter (FACS). (a) Close-up of flow chamber and separation compartment of cell sorter FACSAria (BD Biosciences; see also Fig. 14.8). Up to four fractions may be collected by deflection into tubes at bottom. (b, c) FACSAria used to analyze CD34+ cell enrichment following CliniMACS (Miltenyi Biotech) selection from leukapheresis of peripheral blood analyzed on. (b) Percentage of CD34+ cells, within a total mononuclear cell population, prior to enrichment. (c) Purified cells following CD34+ selection. (Courtesy of Paul O’Gorman Leukaemia Research Centre, Glasgow.)

FIGURE 14.8 Flow Cytometry. Principle of operation of flow cytophotometer. Cell stream in D-PBSAenters at the top, and sheath liquid, also D-PBSA, is injected around the cell stream to generate alaminar flow within the flow chamber. As the cell stream exits the chamber, it cuts a laser beam,and the signal generated triggers the charging electrode, thereby charging the cell stream. The cellstream then breaks up into droplets, induced by the 15-kHz vibration transducer attached to the flowchamber. The droplets carry the charge briefly applied to the exiting cell stream and are deflectedby the electrode plates below the flow chamber. The charge is applied to the plates with a sufficientdelay to allow for the transit time from cutting the beam to entering the space between the plates (seealso Fig. 14.7).