BD Pharmingen™ FITC Mouse Anti-Ki-67 Set

Ki-67 staining protocol by flow cytometry:

1.  Harvest, count and pellet cells following standard procedures (Note: Ki-67 is expressed by the proliferative cells. You may get no staining with the resting cells, e.g. unstimulated PBMC).

2.  While vortexing, add 5 ml drop by drop of cold 70% - 80% ethanol into the cells pellet (1-5 x 10e7 cells). Then incubate at -20°C for 2 hours minimum. These fixed cells can be used up to 60 days after fixing (Store at -20°C).

3.  Add 30-40 ml wash buffer (PBS with 1% FBS, 0.09% NaN3 pH7.2) to the fixed cells. Centrifuge the cells for 10 minutes at 1000 rpm and aspirate supernatant. Wash one more time with 30-40 ml of wash buffer. Centrifuge at 1000 rpm for 10 minutes and aspirate supernatant.

4.  Resuspend the cells to a concentration of 1 X 10e7/ ml (1 X 10e6/100 µl).

5.  Transfer 100 µl cell suspension into each fresh tube.

6.  Add 20 µl of properly diluted antibody 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 PBS washing buffer at 1000 rpm for 5 minutes.

9.  Aspirate the supernatant.

10. For direct conjugated antibody: go to steps 13 & 14.

11. For purified antibody: add 50 µl of diluted secondary antibody at optimal concentration (Cat. No. 555988), incubate at RT for 30 minutes in the dark.

12. Repeat step 8 & 9.

13. Add 0.5 ml of PBS wash buffer into each tube. For FITC conjugated antibody, add 10 µl of PI Staining Solution (Cat. No. 556463); for PE conjugated antibody, add 20 µl BD Via-Probe™ Cell Viability Solution (Cat. No. 555816) into each tube.

14. Analyze the sample with FACS.

1. This antibody has been optimized and preassayed with its matched isotype control to be used at the recommended volume of 20 ul/test. Titration of the reagents or substituting with other (non-matched) isotype control is NOT recommended.
2. Caution: Sodium azide yields highly toxic hydrazoic acid under acidic conditions. Dilute azide compounds in running water before discarding to avoid accumulation of potentially explosive deposits in plumbing.
3. Source of all serum proteins is from USDA inspected abattoirs located in the United States.
4. Species cross-reactivity detected in product development may not have been confirmed on every format and/or application.
5. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
6. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.