cell cycle analysis by dna content - protocols - flow cytometry - uc san diego moores cancer center

8/12/2019 Cell Cycle Analysis by DNA Content - Protocols - Flow Cytometry - UC San Diego Moores Cancer Center

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Cell Cycle Analysis by DNA Content(Propidium Iodide)

Fixation

1. Wash cells by centrifugation (e.g. 200 x g, 5 min, 4C) in protein-free buffer, such as Phosphate

Buffered Saline without Ca+2or Mg+2(PBS).

2. (Optional) Repeat step 1.

3. Resuspend at 2 x 106cells in 1 ml ICE COLD BUFFER. Cell number will effect stainingquality!Optional:Use pre-coated or silanized polypropylene tubes to minimize sticking. Pre-coattubes overnight with 2% Bovine Serum Albumin (BSA) in PBS.

4. Vortex gently, slowly adding the cell suspension dropwise to 9 ml of 70% ethanol in a 15 ml

polypropylene centrifuge tube (Falcon Cat. No. [35]2097). OR: Vortex gently, slowly adding thecell suspension dropwise to an equal volume of COLD ABSOLUTE ethanol. Optional:Observecell preparation with a microcope to verify minimum cell clumping.

5. Store at 4C to - 40C for AT LEAST 2 hours, 12 - 24 hours is best. Can be stored for up to 2years before staining.

6. Centrifuge cells at 200 x g, 10 min, 4C.

7. Resuspend pellet in 3 ml COLD PBS and transfer to Falcon 12 X 75 mm (Cat. No. [35]2054)polystyrene tubes for staining if other tubes (polypropylene) were used for the fixation steps above.Falcon Cat. No. [35]2235 have nylon filter caps and will remove clumps.

Staining with Propidium Iodide (PI)

1. Wash cells at least once with COLD PBS. Cells may form a diffuse ring-shaped pellet, so centrifugelonger ( e.g. 200 x g, 10 min, 4C).

2. Resuspend cells in 300 - 500 l PI/Triton X-100 staining solution: to 10 ml of 0. 1 % (v/v) TritonX-100 (Sigma) in PBS add 2 mg DNAse-free RNAse A (Sigma) and 0.40 ml of 500 g/ml PI(e.g., Roche). Prepare freshly. A stock solution of PI, made by dissolving 1 mg PI in 2 ml water,can be stored several months at 0 to 4C. (Or buy 500 g/ml PI from Roche new Catalog #11348639001, old Cat. No. 1348639). Note:If the RNAse is not DNAse-free, boil a solution of2 mg RNAse A in 1 ml water for 5 min. Aliquot and store at -20C.

3. Incubate 37C for 15 minutes or for 30 min at 20C.

4. Transfer tubes to ice or store at 4C PROTECTED FROM LIGHT.

5. Acquire data on flow cytometer within 48 hours (but might last up to 2 weeks). May require nylonmesh filtration (eg, Filcons, BD Cat. No. 340627) to remove cell clumps or syringing (25 gauge,UCSD Storehouse # 7245) to break up cell clumps. Can acquire 5-30 samples per hour,depending on cell preparation.

6. MulticycleAV (IBM-PC) or ModFit LT (Macintosh) is used to fit the data to various cell cyclemodels. See below for examples

This a screen shot of a typical profile from MulticycleAV:
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Click on this figure to see the full screen.

Here's a ModFit LT output example from the same FCS file:

Click on this figure to see the full screen.

References

1. Shapiro, HM, Practical Flow Cytometry, second edition. New York: Alan R. Liss, Inc; 1988. 353p.

2. Darzynkiewicz, Z, Nucleic Acid Analysis. In: Robinson, JP, managing editor. Current Protocols inCytometry. New York : J Wiley & Sons, Inc; 1997. Chapter 7.

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cell cycle analysis by dna content - protocols - flow cytometry - uc san diego moores cancer center

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