Cell Aggregation Assay see

1. Prewarm aggregation buffer to 37°C (see Note 1).

2. Collect 5.0 x 106 cells in an eppendorf tube.

3. Centrifuge the cells at 300g for 5 min and remove the supernatant.

4. Wash the cell pellet in 1 mL of aggregation buffer and centrifuge again.

5. Using a transfer pipet, vigorously resuspend the cells in 1 mL of aggregation buffer. During initial experiments, visually inspect the cells using a phase microscope to ensure that the cells are in a single-cell suspension.

6. Split the sample into two equal volumes (i.e., 500 ^L).

7. To one tube, add 65 ^L of a heparin solution (see Note 2).

8. To the other tube, add 65 ^L of sterile deionized water.

9. Incubate the cells undisturbed at 37°C for 1 h.

10. Resuspend the cells by gently pipetting the solution 5 times. This is a critical step in that pipetting must be uniformly gentle or aggregates may be disrupted (see Notes 3 and 4).

11. Cells are counted in both the heparin-containing and heparin-free samples using a hemocytometer. A single cell or aggregates containing more than 3 cells are counted as 1. However, in aggregates consisting of 2 or 3 cells, each cell is counted (see Note 5).

Aggregation Cell

Fig. 1. Diagram of the aggregation assay. Cells from each sample are separated into two tubes containing equal numbers of cells. Heparin is added to one tube (to block aggregation), while the other cells are allowed to aggregate. Following gentle pipetting, the number of cells in each tube is determined. Single cells and aggregates containing more than three cells are counted as 1. Cells within groups of 2 or 3 are counted individually.

Fig. 1. Diagram of the aggregation assay. Cells from each sample are separated into two tubes containing equal numbers of cells. Heparin is added to one tube (to block aggregation), while the other cells are allowed to aggregate. Following gentle pipetting, the number of cells in each tube is determined. Single cells and aggregates containing more than three cells are counted as 1. Cells within groups of 2 or 3 are counted individually.

12. The percentage of cells in aggregates can now be determined as follows: total cells - (single cells + aggregates)

x 100 = % cells in aggregates total cells total cells = number of single cells in heparin-containing sample single cells + aggregates = number of single cells plus number of aggregates in the heparin-free sample

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