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DNA Flow Cytometry (Cell Cycle Analysis)

Stock Solution

Trisodium citrate dehydrate Na3C6H5O7.2H2O 柠檬酸三钠    500 mg

Nonidet P40 (NP40)             0.5 ml

Spermine tetrahydrochloride 四氢氯化精胺      261 mg

Tris (hydroxymethyl)-aminomethane (Tris base)     30 mg

2d-H2O                  Total   500 ml

Titrate pH to 7.6, Store at 4℃

Citrate Buffer

Sucrose 蔗糖          42.75 g (250 mM)

Trisodium citrate dehydrate Na3C6H5O7.2H2O     2.88 g (40 mM)

DMSO             25 ml

2d-H2O                 Total   500 ml

Titrate pH to 7.6, Store at 4℃

Solution A:

Trypsin    胰蛋白酶 0.6 mg

Stock soln     100 ml

Titrate pH to 7.6, Store at 4℃

Solution B:

Trypsin inhibitor    胰蛋白酶抑制剂 50 mg

RNase A       10 mg

Stock soln       100 ml

Titrate pH to 7.6, Store at 4℃

Solution C:

Propidium iodide        50 mg

Spermine tetrahydrochloride  116 mg

Stock soln          100 ml

Titrate pH to 7.6, Store at 4℃. Avoid from light exposure

Procedures of DNA Flow Cytometry (Cell Cycle Analysis)

-  Cell pellet (2-5X105 cells/sample)

-  Resuspend and fix cells in cold citrate buffer for 5-10 min at 4℃to make the cell density 2X105 cells/200 ml citrate buffer. (May keep cells at 4℃for up to 3-5 days)

-  Prewarm solutions A and B at room temperature.

-  Transfer cells to a Falcon tube (Becton Dickinson FCM tube, #2052 or #2054).

-  Add 450 ml Soln A for 10 min at room temp. vigorous vortexing.

-  Add 375 ml Soln B for 10 min at room temp. vigorous vortexing.

-  Add 100 ml cold Soln C to stain the nuclei. Brief vortexing.

-  Run FAScan

The method is according to Vindelov, et al. (Cytometry 1983; 3:323-7) with some modifications.