How to Synchronize Cells in Culture

To understand the molecular and biochemical basis of cellular growth and division, you need to study cell cycle regulatory mechanisms. Cell cycle studies mainly involve synchronization or coordination of cells at a specific phase. Cell synchronization is a method of bringing cells at different stages to one particular stage of cell division. There are many methods established by scientists in the laboratory to synchronize cells at specific phases of the cell cycle. The most common methods are serum deprivation of cells for a period of time or use of pharmacological agents such as Nocodazole. Either of these methods brings the cells to stop at a specific phase of cell cycle.

Instructions

  1. Serum Starvation

    • 1

      Plate your cells in regular cell culture medium supplemented with 10 percent fetal bovine serum for 12 to 14 hours and let the cells attach to the tissue culture dishes.

    • 2

      Once the cells have attached, remove the media and rinse the cells with 10 milliliters of phosphate buffered solution.

    • 3

      Add new growth media supplemented with 0.2 percent fetal bovine serum for 16-48 hours. The time period depends on the type of cells you are using. Some cells require less time while others require more time. This stage is the serum starvation period.

    • 4

      After the set time period is over, rinse the cells again with 10 milliliters of phosphate buffered solution.

    • 5

      Replace the old growth media supplemented with 10 percent fetal bovine serum. The cells are now synchronized and are ready for your experiment.

    Nocodazole treatment

    • 6

      Plate your cells in regular cell culture medium supplemented with 10 percent fetal bovine serum for 12-14 hours and let the cells attach to the tissue culture dishes.

    • 7

      Remove the media and rinse the cells with 10 milliliters of phosphate buffered solution.

    • 8

      Add regular growth media supplemented with 10 percent fetal bovine serum and 100ng/ml of nocodazole to the cells for 12 hours in 37 degrees Celsius incubator.

    • 9

      Observe the cells under the microscope after 12 hours of treatment. The cells must be rounding up and getting detached from the culture dish.

    • 10

      Collect the cells by tapping the culture dish and by pipetting the medium up and down several times. Transfer the medium containing cells to a 50-milliliter polypropylene tube.

    • 11

      Centrifuge the cells at 100 x g for 10 minutes. Discard the supernatant. Wash the cells in 10 milliliters of phosphate buffered solution and centrifuge again at 100 x g for 10 minutes.

    • 12

      Suspend the cells in regular growth medium supplemented with 10 percent fetal bovine serum. The cells are ready for your experiment.

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