Principle of primary cell cycle assay and BrdU method
principle:
Cell cycle: The period of time that a cell has passed from one end of a cell division to the end of the next division. The cell cycle reflects the rate of cell proliferation. The periodic measurement of a single cell can be performed by means of time-lapse photography, but it does not represent the cycle of the cell population, so other methods are now used to measure the population cycle.
There are many methods for measuring the cell cycle, such as isotope labeling and cytometry.
BrdU (5-bromodeoxyuridine) can be used as a raw material for DNA replication after adding to the medium. After two cell cycles, the DNA of BrdU containing two single strands in the cell will account for l/2, which is reflected in The chromosome should appear as a single light stain. If three cycles are experienced, about half of the chromosomes are lightly stained with two monomers, and the other half is deep and shallow. If the cells have only experienced one cycle, both monomers are deeply stained. The value of the cell cycle can be calculated by taking the proportion of each phase in the split phase.
Reagent
1, instruments, supplies: with conventional cell culture
2. Reagents: BrdU (1.0mg/ml), methanol, glacial acetic acid, Giemsa dye solution, colchicine, 2×SSC solution
BrdU method 1. When the primary cells grow to the exponential phase, BrdU is added to the culture solution to a final concentration of 10 μg/ml.
2, 44 hours plus colchicine, so that each ml contains 0.1μg.
After 3 and 48 hours, the cells are routinely digested into a centrifuge tube, and the floating cells of the culture supernatant are also collected into a centrifuge tube.
4. Conventional chromosome production.
5, chromosome slides placed on a 56 ° C water bath lid, 2 × SSC solution, UV irradiation 6 minutes from the UV tube for 30 minutes.
6. Discard 2×SSC solution and rinse with running water.
7. The Giemsa solution was stained for 10 minutes, rinsed with water and allowed to dry.
8. Microscopic examination of 100 divisional phases, counting the first, second, third, and fourth cell division indices.
9. Calculation: Cell cycle (Tc) = 48/{(M1+2M2+3M3+4M4)/100} (hours)
Attached:
(1) Preparation of BrdU: BrdU 10mg ten double distilled water 10ml Store at 4°C in the dark.
(2) 2 × SSC preparation: NaCl 1.75 g, trisodium citrate • 2H2O 0.88 g, add water to 100 ml, and store at 4 ° C.
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