Immunomagnetic cell separation can be used to further debulk the mixture of white blood cells obtained from the density gradient separation and thus further enrich the suspension with rare cancer cells (see Chapter 21). Before immunochemical staining, it is usual to concentrate the cells onto polycarbonate membranes so that they are immobilized (see Note 8).
1. Place cell suspension into a syringe (syringe size will depend on the volume recovered).
2. Assemble the membrane holders with the polycarbonate membrane previously moistened with labeling buffer, attach syringes, and mount in a syringe pump. Set flow rate to a maximum of 1 mL/min (see Note 9).
3. While the syringe pump is running, prepare antibody-enzyme and substrate solutions. Usually a dilution of 1:1000 of the antibody-enzyme is enough to successfully stain cells (see Note 10); to prepare substrate, follow manufacturer's recommendations.
4. Once all cell suspension has passed through the filter, detach the membrane holders from the syringe and using a 1-mL syringe, add enough antibody-enzyme solution to cover the membrane. Incubate in the refrigerator (dark, cool place) for about 30 min.
5. Reconnect to the syringe pump and wash the membrane with labeling buffer (again, flow rate not higher than 1 mL/min) and then add enough substrate solution to cover the membrane using a syringe. Incubate for approx 15-20 min. Push liquid out of the membrane holders and recover the membrane. Analyze under light microscope. A picture showing recovered cells is shown in Fig. 2.
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