Notes

1. It is important to avoid confluent cultures in this protocol. Confluent cultures contain few cells growing, and there might be differences in density within the same population. Cultures in growing phase will have a better chance to yield a cell population with a consistent density.

2. The use of trypsin is common to detach cells from adherent cultures; however, in this case it is not recommended. MCF-7 cells treated with trypsin tend to form clumps, which are difficult to breakup. The use of Accutase yields a mostly single-cell suspension that will not form clumps even after it has been washed with labeling buffer. Total cell count and viability assays will also be easier for the researcher to carry out.

3. Determining cell concentration in a buffy coat sample using a particle counter can be difficult. If the laboratory has access to hematological material, it is recommended to use the Unoppette system (Becton Dickinson), after diluting the sample. Results from particle counter and manual counting should not differ by more than 5%.

4. The seeding of carcinoma cells must be performed based on a live, healthy cell count. Dead cells (those that stained with Trypan blue) are not to be considered because they are expected to sediment in the high-density region.

5. This protocol mentions a 0.1% of rare cells seeded into the buffy coat. It is possible to increase or reduce the percentage of cells. However, less than 0.1% of cells might make it difficult to obtain reliable recovery data, and a larger percentage would not be a good representation of "rare" cells circulating in blood.

Fig. 2. Photographs of MCF-7 cells recovered from the enriched fraction after immunomagnetic sorting and stained with anti-HEA FITC as primary antibody anti-FITC phosphatase alkaline as secondary antibody and enzyme substrate (FastRed™). In this image, cells appear as medium-grey bodies. In specimens viewed in the laboratory, the cell bodies would appear stained red as opposed to grey. The dark, defined circles are pores in the membrane.

Fig. 2. Photographs of MCF-7 cells recovered from the enriched fraction after immunomagnetic sorting and stained with anti-HEA FITC as primary antibody anti-FITC phosphatase alkaline as secondary antibody and enzyme substrate (FastRed™). In this image, cells appear as medium-grey bodies. In specimens viewed in the laboratory, the cell bodies would appear stained red as opposed to grey. The dark, defined circles are pores in the membrane.

6. It is very important to maintain density gradient and cell suspension at room temperature before and during centrifugation. Temperature affects density and therefore the separation and yield.

7. Depending on the number of cells used in the centrifugation step, there will be a variable cell concentration in the tubes to test. Dilutions may be necessary.

8. To carry out immunomagnetic cell separation, cells will be stained with microbeads that target the CD45 receptor. At the same time, cancer cells must have been labeled with anti-HEA FITC.

9. In any case do not exceed 1 mL/min flow rate for the syringe pump. Higher flow rates could damage recovered cells because they could have been squeezed into the 5-^m pore size of the membrane. However, filtration through this pore size will eliminate some remaining erythrocytes and debris present in the sample.

10. Test every lot of antibody received; the best results are obtained with a titrated antibody response.

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