Cell synchronisation Methods

Introduction

Cell synchronisation is a process by which cells at different stages of the cell cycle in a culture are brought to the same phase. Cell synchronisation is used to study the progression of cells through the cell cycle. Cells can be synchornised and separate in 2 ways.

  1. Chemical Blockade
  2. Physical Fractionation

Chemical Blockade

As the names suggests this method uses chemicals such as Thymidine to block metabolic reactions. This can be achieved through inhibition of DNA synthesis largely during the S-Phase of the cell cycle. Inhibitors such as thymidine, aminopterin, hydroxyurea and cytosine arabinoside can have variable effects. Serum starving your cells for 24 hrs will result in an accumulation of cells at G1 phase. The effects of serum starvation can be reversed by the addition of serum to media once cell synchrony has occured.

Physical Fractionation

Physical fractionation or cell separation techniques can be based on cell density, cell size, affinity of antibodies on cell surface epitopes and light scatter or fluorescent emission by labeled cells.

Centrifugal separation or fluoresecene-activated cell sorting are primarily used for physical fractionation. Centrifugal separation enables the separation of cells based on size and sedimentation velocity. Fluorescence-activated cell sorting (FACS) sorts cells on based on differences which can be detected by light scatter (e.g. cell size) or fluorescence emission (by penetrated DNA, RNA, proteins, antigens). This can be carried out using a flow cytometer or fluorescence-activated cell sorter.

Stages of the Cell Cycle

The cell cycle exists as 3 main stages:

  • G0/G1 phase – cell rest and recovery in preparation for subsequent rounds of cell division
  • S Phase – DNA replication (interphase)
  • G2/M phase – chromosome segregation and mitosis

The below sample protocols are optimised and focus on cell synchronisation by chemical blockade. As Mentioned previously different chemicals can induce cell synchronisation at different stages in the cell cycle (see below table).

Cell Cycle Stage Targeted Treatment used
G1 arrest Double Thymidine block,Serum starvation. Inhibition of cyclin dependent kinase (CDKs)
G2 arrest Inhibition of microtubules, Inhibition of cyclin dependent kinase (CDKs)
G2/M arrest RO-3306
M-Phase Taxol, Nocodazole

Table 1. Cell synchronisation targets and treatments

Cell synchronisation through double thymidine block

  1. To synchronise cells at the G1/S border a freshly prepared thymidine solution (16 mM) was made up in complete medium and filter-sterilised using a 0.22 µm filter disc.
  2. Cells (1 X 107) were cultured in 60 ml complete medium containing thymidine (2 mM) for 16 h in a 175 cm3cell culture flask.
  3. The cells were then resuspended in complete medium (52.5 ml) for 8 h to allow cells to reenter the cell cycle.
  4. Cells were harvested by centrifugation at 400 x g for 5 min and washed X 2 in complete medium (10 ml). Cells (1 X 107) were cultured in 60 ml complete medium containing thymidine (2mM) for 16 h to synchronise cells at the G1/S border.
  5. Cell synchronisation was confirmed by flow cytometry.

Cell synchronisation by Nocodazole treatment

  1. Cells (1 x 107) were synchronised in mitosis by treating cells for 18 h with Nocodazole (100 nM) and incubated at 37 °C in a 175 cm2
  2. Cells were washed with complete medium X3 and re-plated in complete medium and samples were taken at 0, 30, 60, 120 and 180 min post-release. A
  3. s a control, one sample was re-plated in the presence of Nocodazole (100 nM) to maintain cells in mitosisand a sample was take 180 min post-release.
  4. Cells were lysed in RIPA buffer and whole cell extracts were prepared. Samples were stored at -20 °C.

The treatment of mitotically synchronised cells with Cdk inhibitors

  1. K562 cells were arrested in G1/S phase by double thymidine block (DTB) as previously described (see first sample protocol)
  2. Cells were harvested by centrifugation at 400 x g for 5 min and washed X 2 in complete medium (10 ml).
  3. The cells (1 x 106/ ml) were then resuspended in complete medium and allowed to rest for 1 h.
  4. Cells were treated with either vehicle (0.1% (v/v) DMSO) or Taxol (1 µM) for 11 h. 12 h post DTB cells were treated with either vehicle (0.1% (v/v) DMSO) or with the Kinase inhibitors, BI2536 (10 µM) Roscovitine (20 µM), RO-3306 (9 µM), Purvalanol A (10 µM)  the proteasome inhibitor, MG-132 (10 µM) for 2 h.
  5. Cells were lysed with RIPA buffer and whole cell extracts were prepared.
  6. Samples were stored at -20 °C.

M-phase treatment of Taxol arrested cells with Cdk inhibitors

  1. Cells (1 x 106) were treated with either Vehicle (0.1% (v/v) DMSO), Taxol (1 µM) or Nocodazole (1 µM) for 12 h.
  2. The cells were treated with the kinase inhibitor Roscovitine (20 µM) or Roscovitine plus the proteasome inhibitor, MG-132 (10 µM) for 2 h. Cells were harvested by centrifugation at 400 x g for  5 min and lysed with 30 µl RIPA buffer.
  3. Samples were stored at -20 °C.

RO-3306-induced G2/M arrest of K562 cells

  1. K562 cells were treated with either vehicle (0.1% (v/v) DMSO) or RO-3306 (9 µM) for 18 h.
  2. Cells were washed X 3 in complete medium and re-plated in 10 ml of complete medium treated with either vehicle (0.1% (v/v) DMSO) or MG-132 (10 µM).
  3. Samples were taken at 0, 15, 30, 45, 60 and 90 min postwash.
  4. K562 cells were lysed with RIPA buffer and whole cell extracts were prepared.
  5. Samples were stored at -20 °C.

Confirmation of cell Synchronisation

Cell synchronisation can be confirmed by microscopy or flow cytometry. Microscopy allows you to see what is actually going on inside you cells. Flow cytometry enables you to compare your treated synchronised cells against a asynchronous control. Briefly the protocol is as follows;

  1. Fix and permeabilize your cells in 70 % ethanol
  2. Stain with 40 µg/ml propidium iodide, and include 25 µg/ml of RNase (to degrade RNA and ensure that you stain DNA only).
  3. Run your samples on the flow cytometer.

 

Icons made by “http://www.freepik.com” title=”Freepik”>Freepik from =”http://www.flaticon.com”

Leave a Reply

Your email address will not be published. Required fields are marked *