Density Gradient Separation

1. Dilute the cell suspension (buffy coat spiked with the cancer cells) with HBSS at different ratios prior to centrifugation. Suggested ratios range from 1:1 to 1:5, cell suspension:HBSS.

2. Mix the suspension thoroughly and proceed to determine the hema-tocrit of the suspension by using the capillary tube method.

3. The density gradient separation protocol was first developed for 50-mL conical tubes but any tube size can be used as long as the amounts used are scaled accordingly. For 50-mL conical tubes, place 18 mL of the density gradient cushion (Accuprep) into the tube, and then lay 22 mL of the cell suspension on top of the gradient cushion. Care should be taken to not mix the cell suspension with the gradient cushion. Centrifuge the tubes for 30 min at 350g at room temperature with the centrifuge brake off (see Note 6).

4. Discard the plasma layer carefully by aspiration using a pipet leaving about 1 cm of plasma on top of the mononuclear cell layer.

5. Using a sterile pipet, collect the cell layer, trying to remove all of the layer as a single aspirate while avoiding contamination from the polymorphonuclear cell layer. It is recommended to wash recovered cells at least once with labeling buffer by centrifugation at 300g.

6. Resuspend the cell pellet in labeling buffer (about 5 mL final total volume) and proceed to count recovered cells in triplicate (see Note 7). An automated particle counter is highly recommended because it is possible to set size gates for cancer cells, erythrocytes, and leukocytes.

7. Plot a graph of percentage recovery vs hematocrit; an example of the graph obtained is shown in Fig. 1. The data show a peak in recovery at approx 22% hematocrit; this value should be used for further experiments.

Hematocrit (% total solids)

Hematocrit (% total solids)

Fig. 1. Recovery of MCF-7 cells seeded at a concentration of 0.13% in buffy coat samples as a function of hematocrit. Samples were loaded on a Ficoll-Hypaque gradient at the concentration specified by the experimental point.

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