Encyclopedia of Experiments: Cancer Research
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Transcript
- Start with a leukemia cell suspension containing the desired cell density. Centrifuge and resuspend the cells in a culture medium containing serum. Serum addition promotes continuous cell growth and cells move across the different cell cycle phases. Dilute the cells in a bromodeoxyuridine, or BrdU, labeling solution and grow them under physiological conditions for the required duration.
Cells in the synthesis or S-phase take up BrdU-- a thymidine analog-- that replaces thymidine during the DNA replication process. Centrifuge the cells to remove excess BrdU and resuspend the cells in a suitable media. Transfer the cells to a multiple well plate. Allow the cells to grow under physiological conditions for different time periods.
At various time periods, cells progress through the remainder of the S, G2, mitosis, and G1 phases and enter into the next cell cycle, allowing for the estimation of cell proliferation. Perform antibody staining to tag BrdU with fluorochrome-antibody and measure cell proliferation using flow cytometry.
Inside the flow cytometer, fluorochromes absorb the laser energy and fluoresce. The number of proliferating cells directly correlates to the fluorescence observed. In the example protocol, we will demonstrate the pulse labeling of acute lymphoblastic leukemia cells.
- This protocol uses the acute lymphoblastic leukemia cell line, NALM6, but can be applied to any non-adherent cell line. Maintain the NALM6 cells in T-75 culture flasks in complete RPMI at 37 degrees Celsius in 5% carbon dioxide in air. After collecting the cells and performing a cell count, resuspend the cells in complete RPMI at 2 times 10 to the 6 cells per milliliter.
Dilute the cells 1 in 2 with BrdU Complete RPMI to produce a final cell concentration of 1 times 10 to the 6 cells per milliliter. Handle BrdU with care as it is a potential mutagen and teratogen. Incubate at 37 degrees Celsius with 5% carbon dioxide for 45 minutes.
Next, dilute the cells 1 in 10 with Complete RPMI. Centrifuge at 150 times G for five minutes and carefully discard all of the supernatant. Resuspend the cells in a small volume of Complete RPMI, perform a cell count, and adjust the cell concentration to 1 times 10 to the 6 cells per milliliter. Pipette 1 milliliter of cells into each well of a 48-well plate.
Pipette 1 milliliter of DPBS into any unoccupied wells to obtain more reproducible results. Incubate at 37 degrees Celsius in 5% carbon dioxide in air for the desired time points, depending on what the experimental design aims to measure. When the incubation is complete, transfer all the cells into FACS tubes.
Rinse each well sequentially with 1 milliliter volumes of PBS to a final total volume of 5 milliliters. Centrifuge at 150 times G for five minutes and carefully remove all the supernatant. The cells are now ready for staining, which should be performed immediately. If surface staining is needed, it should be performed now, prior to fixation.
