Encyclopedia of Experiments: Cancer Research
A subscription to JoVE is required to view this content.
Transcript
- To begin, add trypsin to a plate containing leukemia cells co-cultured with bone marrow stromal cells for cell dissociation. Centrifuge and re-suspend the pellet using pre-warmed media to maintain cell viability. The G-10 column contains cross-linked dextran beads that swell in water to form a gel matrix.
Equilibrate the G-10 column with pre-warmed media to fill the space between beads, ensuring slow elusion of the media from the column. Maintain a suitable temperature in the column to prevent the formation of air bubbles. Load the cell mixture slowly onto the gel beads in the G-10 column. Incubate the cells on the G-10 column at room temperature for the desired duration. Based on cell surface characteristics and size, the gel beads preferentially restrict the larger bone marrow stromal cells, allowing the separation of the leukemia cells.
Add pre-warmed media to the column and collect the suspension of leukemia cells. Centrifuge the cell suspension and resuspend the pellet in an appropriate buffer. Use the pure population of leukemia cells for further analysis. In the following example, we will separate a pure population of leukemia cells from stromal co-culture using the G-10 column.
- With the stopcock completely closed for each sub population, use a 1000 microliter pipette to add the entire 1 milliliter volume of cells, drop wise, onto the top of one of the prepared G-10 columns, making sure the cells remain on top of or within the G-10 pellet. Allow the cells to incubate on the G-10 pellet for 20 minutes at room temperature. Then, add 1 to 3 milliliters of fresh, pre-warmed medium to the column and open the stopcock so that the medium slowly exits the columns drop wise.
Continue to add pre-warmed medium to the columns in 1 to 2 milliliter increments. When a total of 15 to 20 milliliters of leukemic cells has been collected, close the stopcock and cap the collection tube. Then, spin down the cells and resuspend the pellet in the appropriate downstream analysis buffer.
