细胞生物学-Cell synchronization

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Cell Synchronization is a process by which cells at different stages of the cell cycle in a culture are brought to the same phase.[1] "Cell synchrony" is required to study the progression of cells through the cell cycle. The types of synchronizations are broadly categorized into two groups: "Physical Fractionation" and "Chemical Blockade."

1.       Cell separation by physical means

Physical fractionation or cell separation techniques, based on the following characteristics are in use: Cell density; Cell size; Affinity of antibodies on cell surface epitopes; Light scatter or fluorescent emission by labeled cells.

The two commonly used techniques are:

(1)Centrifugal separation

The physical characteristics — cell size and sedimentation velocity — are operative in the technique of centrifugal elutriation. Centrifugal elutriator (from Beckman) is an advanced device for increasing the sedimentation rate so that the yield and resolution of cells is better. The cell separation is carried out in a specially designed centrifuge and rotor.

(2)Fluorescence-activated cell sorting

Fluorescence-activated cell sorting (FACS) is a technique for sorting out the cells based on the differences that can be detected by light scatter (eg. cell size) or fluorescence emission (by penetrated DNA, RNA, proteins, antigens). The procedure involves passing of a single stream of cells through a laser beam so that the scattered light from the cells can be detected and recorded. There are two instruments in use based on its principle:

    a) Flow cytometer

    b) Fluorescence-activated cell sorter

2.       Cell separation by chemical blockade

The cells can be separated by blocking metabolic reactions.[2] Two types of metabolic blockades are in use:

(1)Inhibition of DNA synthesis

During the S phase of cell cycle, DNA synthesis can be inhibited by using inhibitors such as thymidine, aminopterin, hydroxyurea and cytosine arabinoside. The effects of these inhibitors are variable. The cell cycle is predominantly blocked in S phase that results in viable cells.

(2)Nutritional deprivation

Elimination of serum from the culture medium for about 24 hours results in the accumulation of cells at G1 phase. This effect of nutritional deprivation can be restored by their addition by which time the cell synchrony occurs.

Ref:

   1.Merrill GF (1998). "Cell synchronization.". Methods in Cell Biology 57: 229–249. doi:10.1016/S0091-679X(08)61582-4. PMID 9648108.

   2.Davis PK, Ho A, Dowdy SF (2001). "Biological methods for cell-cycle synchronization of mammalian cells". BioTechniques 30 (6): 1322–1331. PMID 11414226.

Attached

Protocol 1

Metaphase Block for Cell Synchronization

Posted on Monday, October 27, 2003
Description
Metaphase Block for Cell Synchronization
Procedure
1. Remove the medium from an exponentially-growing cell culture and rinse it with 10 ml of PBS. (See Hint #1)
2. Replace the PBS with Complete Medium with Nocodazole and allow the culture to incubate at 37°C for 12 hr.
3. Shake off loosely attached, rounded mitotic cells by gently knocking the plate and pipetting medium over the cell layer a few times.
4. Collect the cell-containing medium and place it in a sterile 50 ml polypropylene tube and pellet the cells by centrifugation at 1000 X g for 10 min. Remove the supernatant (the cell pellet may be small) and resuspend the cells in 15 ml of PBS.
5. Repellet the cells at 1000 X g for 10 min and remove the PBS. Resuspend the cells in complete medium and proceed with experiment or return the cells to cell culture flask and incubate at 37°C.
Recipes
PBS pH 7.2
2.7 mM KCl
4.3 mM Sodium Phosphate Dibasic (Na2HPO4)
1.8 mM Potassium Phosphate Monobasic (KH2PO4)
137 mM NaCl

Complete Medium 100 Units/ml Penicillin
100 ìg/ml Streptomycin
DMEM
10% (v/v) Calf Serum

Nocodazole Stock (10,000 X) 6 g/ml in DMSO (CAUTION See Hint #2)


Complete Medium with Nocodazole 100 Units/ml Penicillin
Dulbecco's Modified Eagle's Medium (DMEM)
100 ìg/ml Streptomycin
10% (v/v) Calf Serum
600 ng/ml Nocodazole (diluted from Nocodazole stock)

Supplies
Tips
1. Different cell lines use different media, thus "complete medium" may be different for your cell line than what is described here.

 

Protocol 2


Cell synchronization using a double thymidine block


1. Cells are grown to a subconfluent density (mid-log phase) in serum-rich medium.

2. Thymidine is added to 2 mM final and incubated for 16 hours for HEp-2, 19 h for HeLa.

3. Cells are washed three times with PBS on plates and refed with fresh serum-rich medium.

4. Incubate for 10 hours before adding 2 mM thymidine.

5. Incubate for 14 hours (17 for HeLa) and wash as described in step 3, refeed, and begin time points.

Note: Conditions for synchronization of different cell lines may vary (e.g. time of incubation).
From
http://elledgelab.bwh.harvard.edu/protocols/mamCell/cellsyn.html

TAG: 细胞生物学

 

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