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 10 7 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% NaN 3), centrifuge for 10 minutes at 200 x g.
4. Resuspend the cells to a concentration of 1 x 10 7/ml.
5. Transfer 100 µl (1 x 10 6 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].