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Flow Cytometry Staining Protocol for Detection of Ki-67

  1. Harvest, count and pellet cells following standard procedures.

    Note: Ki-67 is expressed by proliferating cells. Using resting cells (eg, unstimulated PBMC) may give negative results.

  2. While vortexing, add 5 ml cold 70% - 80% ethanol dropwise into the cell pellet (1-5 x 107 cells). Incubate at -20°C for at least 2 hours. These fixed cells can be stored at -20°C for up to 60 days prior to staining.
  3. Wash twice with 30-40 ml staining buffer (PBS with 1% FBS, 0.09% NaN3), centrifuge for 10 minutes at 200 x g.
  4. Resuspend the cells to a concentration of 1 x 107/ml.
  5. Transfer 100 µl (1 x 106 cells) cell suspension into each sample tube.
  6. Add 20 µl of properly diluted anti-Ki-67 antibody (clone B56) according to the protocol into the tubes above. Mix gently.
  7. Incubate the tubes at room temperature (RT) for 20-30 minutes in the dark.
  8. Wash with 2 ml of staining buffer at 200 x g for 5 minutes.
  9. Aspirate the supernatant.
  10. If using directly conjugated anti-Ki-67 (PE set: cat. no. 556027, FITC set: cat. no. 556026), proceed to step 13.
  11. If using purified anti-Ki-67 (cat. no. 556003), add 50 µl of diluted secondary antibody (eg, cat. no. 555988) to each sample tube and incubate at RT for 30 minutes in the dark.
  12. Repeat steps 8 & 9.
  13. Add 0.5 ml of staining buffer to each tube. If using FITC conjugated anti-Ki-67 or secondary antibody, add 10 µl of Propidium Iodide Staining Solution (cat. no. 556463) to each tube; for PE conjugated anti-Ki-67 or secondary antibody, add 20 µl BD Via-Probe™ Cell Viability Solution (cat. no. 555816) to each tube.
  14. Proceed to flow cytometric analysis.

Note: This protocol is also suitable for staining of a variety of other intracellular molecules, such as p53, PCNA, or p16[INK4].