BrdU/PI Staining for Cell Cycle Protocol

For 3-6X10^6 cells

Labelling and Fixing the Cells:

1. Incubate the cells with 20µM BrdU
   • 15-30 min (depending on how fast the cells are growing) before the time of harvest, add BrdU to the medium in the dishes to a final concentration of 20µM (e.g., 2ul 10mM stock per ml medium).
   • Put the cells back into the incubator for 15-30 min.
2. Fix the cells with cold 95% ethanol
   • Harvest the cells as usual (PBS-EDTA or trypsinization, if attached) and chill to 4°C.
   • Transfer the cells into 12x75 mm Falcon 6-ml (2058 or 2008) tubes (the same ones used for the flow cytometer).
   • Pellet cells at 4°C for 5 min at 300xg (between 1000-1500 rpm on most tabletop refrigerated centrifuges). Aspirate supernatant.
   • Resuspend cells in 200 µl ice-cold PBS=+EDTA. Place tubes in ice bath.
   • Quickly add 1.8 ml ice-cold 95% EtOH and immediately mix by swirling the tube by hand. Another way is to add the EtOH slowly while gently vortexing (speed 1 or on "shaking" setting). Note: EtOH-fixed cells are fragile! Treat them gently.
   • Let them sit on ice for at least 45 min. After this, the cells may be stored at 4°C overnight or -20°C for 2-4 weeks before staining.

Releasing the Nuclei and Removing RNA:

1. Prepare fresh solutions of pepsin and RNase A. (See end for details.)
2. Dilute fixative and pellet cells.
   • Add 2 ml PBS= and pellet cells at room temp for 5 min at 300xg. Aspirate supernatant.
   • Resuspend cells in 2 ml PBS= and pellet cells at room temp for 5 min at 300xg. Aspirate supernatant.
3. Treat with pepsin and HCl. Neutralize acid.
   • Resuspend pellet in 0.5 ml pepsin/HCl solution. Incubate in the dark for 30 min at room temp (This digests plasma membrane and releases nuclei).
   • Pellet nuclei at room temp for 5 min at 600xg (between 1500-2000 rpm on most tabletop regfrigerated centrifuges). Aspirate supernatant. Note: The nuclei pellet is much smaller than the cell pellet, and thus may be very hard to see or even invisible. Be extra-careful when aspirating supernatant to avoid sucking out the pellet.
   • Gently resuspend nuclei in 1 ml 2N HCl.
   • Incubate in the dark for 30 min at 37°C, shaking tubes every 10 min.
   • Add 2 ml 0.1M sodium tetraborate, gently vortex for 10 sec to mix well.
   • Pellet nuclei at room temp for 10 min at 600xg. Aspirate supernatant.
   • Wash pellet twice by resuspending in 3 ml PBS-TB, pelleting at room temp for 10 min at 600xg, and aspirating supernatant. Note: Tween-20 keeps nuclei from clumping and BSA helps prevent DNA from clumping.
4. Treat with RNase A.
   • Resuspend pellet in 1 ml RNase A (10µg/ml) in PBS-TB. And incubate in the dark for 30 min at 37°C.
   • Pellet nuclei at room temp for 5 min at 600xg. Aspirate supernatant.
   • Resuspend pellet in 3 ml PBS-TB and pellet at room temp for 5 min at 600xg.
   • Carefully aspirate all of the supernatant.

Staining with Anti-BrdU and PI:

1. Prepare a fresh 1:50 dilution of goat anti-mouse IgG-FITC in PBS-TG.
2. Stain with Mouse Anti-BrdU.
   • Resuspend pellet in 50 µl PBS-TB.
   • Add 20 µl anti-BrdU antibody, and triturate several times to mix well. Note: Use a new pipette tip for each tube. Make sure there is enough antibody for all of the samples; do not switch lots mid-experiment.
   • Incubate in the dark for 90 min at room temp or overnight at 4°C.
   • Add 3 ml PBS-TB to each tube and pellet nuclei at room temp for 10 min at 600xg. Aspirate supernatant.
3. Stain with GAM-FITC.
   • Resuspend pellet in 200 µl 1:50 GAM-FITC in PBS-TG.
   • Incubate in the dark for 30 min at room temp.
   • Add 3 ml PBS-TB to each tube and pellet nuclei at room temp for 10 min at 600xg. Aspirate supernatant.
   • Resuspend pellet in 3 ml PBS-TB and pellet nuclei at room temp for 10 min at 600xg. Aspirate supernatant.
4. Stain with propidium iodide (PI).
   • Resuspend pellet in 0.5 ml PI (50µg/ml) in PBS-TB.
   • Incubate in the dark overnight at 4°C. Note: Samples should be analyzed within 48 hours of PI staining.
   • Filter samples through 40µm nylon mesh (e.g., Nitex) immediately before analyzing on flow cytometer.

Solutions:

Prepare fresh on the day of use:

• Pepsin (0.04% (w/v)): 0.4mg/ml in 0.1 N HCl.
• Anti-mouse IgG-FITC: dilute 1:50 in PBS-TG
• RNase A (10µg/ml): dilute 1mg/ml stock RNase A to 10µg/ml in PBS-TB.

Stock solutions stored frozen in aliquots:

• BrdU (10mM): 3.071mg/ml in double-distilled water
• RNase A: 1 mg/ml in PBS, heated at 95°C for 20-25min, then allowed to cool slowly to room temp (turn off water bath and leave tube in it).
• PBS-TG: PBS + 0.5% (v/v) Tween-20 + 0.1% goat serum

Solutions stored at 4°C:

• PBS
• PBS-TB: PBS + 0.5% (v/v) Tween-20 + 0.1% (w/v) BSA [4°C]
• PI stock (10mg/ml) in 95% EtOH
• PI Stain: 50µg/ml in PBS-TB

Solutions stored at room temp:

• 0.1N HCl
• 0.1 M Sodium Tetraborate (NaB4O7)