Cell Cycle Staining Protocol

Materials

  • Cell Cycle Kit (Beckman Coulter, Part Number C03551)*
  • PerFix-nc Buffer 1 (Beckman Coulter, Part Number B10825)
  • Cyclin A2-FITC
  • Fetal Calf Serum (FCS)
  • Phosphate Buffered Saline (PBS)
  • Phospho-Histone H3 Rabbit mAB-A647
  • CytoFLEX Flow Cytometer equipped with a Blue and Red Laser (Beckman Coulter)*
  • Kaluza Analysis Software version 2.1 or greater (Beckman Coulter)*

Protocol

Sample Fixation (recommended procedure)

  1. Harvest the cells.
  2. Add 1x106 cells in a 5 mL tube.
  3. Centrifuge the tube at 300 x g for 5 minutes; aspirate the supernatant.
  4. Re-suspend the pellet in 50 μL undiluted FCS.
  5. Add 2.5 μl PerFix-nc Buffer 1, vortex immediately and incubate for 15 minutes at 18-25 °C.
  6. Add 3 mL of PBS.
  7. Centrifuge the tube at 300 x g for 5 minutes; aspirate the supernatant.
  8. Re-suspend the pellet in 3 mL PBS.
  9. Centrifuge the tube at 300 x g for 5 minutes; aspirate the supernatant.
  10. Proceed with staining procedure.

Staining procedure: Cell Cycle Staining

  1. Re-suspend the pellet of fixed cells in 0.5 mL cell cycle kit, vortex the tube and incubate for 15 minutes protected from direct light exposure to light, between 18 and 25 °C.
  2. The sample is ready for acquisition (see Measure Cell Fluorescence by Flow Cytometry).

Measure Cell Fluorescence by Flow Cytometry

  1. Set up and adjust the CytoFLEX flow cytometer with a blue laser (488 nm) and detection filters 610/20 nm bandpass for PI. For multivariate analysis, set up and adjust the CytoFLEX flow cytometer with a blue laser (488 nm) and detection filters 610/20 nm bandpass for PI and 525/40 nm bandpass for FITC and the red laser (638 nm) and detection filter 660/10 nm bandpass for Alexa Fluor™ 647.
  2. Set the flow rate at low flow rate to ensure accuracy.
  3. Add a plot with PI versus Time to monitor the stability of the flow rate. Gate out any perturbations in the flow for subsequent analysis if needed.
  4. Add a dot plot PI-Area versus PI-Hight to gate on singlet events. Singlet events are presented in a diagonal pattern. Doublets will have higher Width values as it takes longer for them to traverse the laser line.
  5. Adjust the gains on the PI channel until the G1 peak MFI is at 2 x 106. This will ensure that the peak for the G2/M stays on scale and that there is good resolution between G2/M and the G1 peaks.
  6. Add the tube and record the acquisition.
  7. When all samples are recorded, save the FCS files and open or import into the analysis software.

*For Research Use Only. Not for use in diagnostic procedures.