Cell cycle analysis of desynchronized cell populations
Cell cycle analysis of desynchronized cell populations
Information on cell cycle dynamics can be obtained by labeling 5' BrdUrd, which can be incorporated into the thymidine site of DNA. Detection of BrdUrd in DNA allows for the counting of S-phase cells, and if specimens are collected at different time intervals, information on cell cycle dynamics can be obtained.
Principle
The basic principle of cell cycle analysis of desynchronized cell populations is to incubate cells continuously in bromodeoxyuridine (BrdUrd) solution, which can be incorporated into the cells during DNA synthesis. At this interval of DNA synthesis, the cells are harvested and the nuclei are stained with bisbenzimidazole, (Hoechst 33258) as well as iodide (PI). On a flow cytometer, these dyes can be excited by UV light to emit blue and red fluorescence. br-dUrd quenches the blue fluorescence of the Hoechst dye. The degree of quenching records the process(s) by which the cell goes through S-phase; the red fluorescence (PI) is present throughout the cell division cycle, and in this way, the entire course of the cell cycle can be tracked.
Operation method
Cell cycle analysis of desynchronized cell populations
Principle
The basic principle of cell cycle analysis of desynchronized cell populations is to incubate cells continuously in bromodeoxyuridine (BrdUrd) solution, which can be incorporated into the cells during DNA synthesis. At this interval of DNA synthesis, the cells are harvested and the nuclei are stained with bisbenzimidazole, (Hoechst 33258) as well as iodide (PI). On a flow cytometer, these dyes can be excited by UV light to emit blue and red fluorescence. br-dUrd quenches the blue fluorescence of the Hoechst dye. The degree of quenching records the process(s) by which the cell goes through S-phase; the red fluorescence (PI) is present throughout the cell division cycle, and in this way, the entire course of the cell cycle can be tracked.
Materials and Instruments
Equipment: Move The basic process of cell cycle analysis of desynchronized cell populations can be divided into the following steps: 1.1 Treat the cells with irradiation, dosing, heat shock, etc., if desired. 1.2 Immediately after treatment, add the appropriate concentration of BrdUrd to the cell culture. 1.3 At fixed time intervals (3~8 h, depending on the cell cycle), harvest some cells. 1.4 Centrifuge cells and resuspend in 500 μl of ice-cold staining solution. Mix briefly by vortexing and leave on ice for 15 min. 1.5 Add 10 μl of PI solution, vortex briefly and mix well, place on ice. 1.6 Analyze on flow cytometer and record red (PI/DNA) and blue (Hoechst/DNA) fluorescence. Adjust the flow rate to 500 cells/sec. If possible, do a waveform analysis of the red fluorescence signal and set a gate to exclude any aggregated adherent nuclei.
① Flow cytometer.
② Centrifuge
Reagents:
① 5'-Bromo-2'-deoxyuridine nucleoside (BrdUrd) (Sigma, St. Louis, MO, catalog no. B5002): make a 10 mM storage solution and freeze.
② Hoechst 33258 (Sigma, catalog number: B2883 or Molecular probes, Eugene, OR, catalog number: H-1398).
③ PI (Sigma, catalog no. P4170 or Molecular probes, catalog no. P-1304): make a storage solution 100 μg/ml in double-distilled water.
④ Staining solution: 100 mM Tris-HCI, pH 7.4, 154 mM NaCl, 1 mM CaCl
2
④ Staining solution: 100 mM Tris-HCI, pH 7.4, 154 mM NaCl, 1 mM CaCl
2
, 0.1% (v/v) Nonidet-P40, 0.2% (w/v) bovine serum albumin (BSA) and 1.2 μg/ml Hoechst 33258.
Cells can be identified by red (PI) and blue (Hoechst) fluorescence at time point 0 and at various phases of the cell cycle, such as G1, S and G2/M phases. When cells were incubated in 40 μM BrdUrd, red fluorescence was enhanced but blue fluorescence was unchanged when cells went through S phase (blue fluorescence was partially quenched by BrdUrd). After 4 h of BrdUrd treatment, the red fluorescence showed a slight bowing pattern in the S phase on the fluorescence graph; at 8 h, the bowing became more pronounced; of course, many cells in the G2/M phase started to divide into the G1 phase (not labeled); at 16 h, at 0 h, all cells in the S phase had reached the G2 phase by this time (labeled as G2 ∗) and a small number of them were dividing ( G1 ∗). Some cells that were in G1 phase at 0 h were now in S phase (Sf); some even entered G2/M ( G2f ). By 28 h, some cells that were in G2 phase at the beginning of the experiment complete the cell cycle and return to G1 phase (labeled G1 ').
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