Each lot of this antibody is quality control tested by intracellular immunofluorescent staining with flow cytometric analysis. For flow cytometric staining, the suggested use of this reagent is 5 ?L per million cells in 100 ?L staining volume or 5 ?L per 100 ?L of whole blood. It is recommended that the reagent be titrated for optimal performance for each application.

Brilliant Violet 750? excites at 405 nm and emits at 750 nm. The bandpass filter 780/60 nm is recommended for detection, although filter optimization may be required depending on other fluorophores used. Be sure to verify that your cytometer configuration and software setup are appropriate for detecting this channel. Refer to your instrument manual or manufacturer for support. Brilliant Violet 750? is a trademark of Sirigen Group Ltd.

Additional reported applications (for the relevant formats) include: immunohistochemical staining of frozen tissue sections¹, Western blotting³, and immunofluorescence microscopy⁴.

Ki-67 Staining Protocol:

1. Prepare 70% ethanol and chill at -20°C.
2. Prepare target cells of interest and wash 2X with PBS by centrifuge at 350xg for 5 minutes.
3. Discard supernatant and loosen the cell pellet by vortexing.
4. Add 3 ml cold 70% ethanol drop by drop to the cell pellet while vortexing.
5. Continue vortexing for 30 seconds and then incubate at -20°C for 1 hour.
6. Wash 3X with BioLegend Cell Staining Buffer and then resuspend the cells at the concentration of 0.5-10 x 10⁶/ml.
7. Mix 100 ?l cell suspension with proper fluorochrome-conjugated Ki-67 antibody and incubate at room temperature in the dark for 30 minutes.
8. Wash 2X with BioLegend Cell Staining Buffer and then resuspend in 0.5 ml cell staining buffer for flow cytometric analysis.

Brilliant Violet 750? excites at 405 nm and emits at 750 nm. The bandpass filter 780/60 nm is recommended for detection, although filter optimization may be required depending on other fluorophores used. Be sure to verify that your cytometer configuration and software setup are appropriate for detecting this channel. Refer to your instrument manual or manufacturer for support. Brilliant Violet 750? is a trademark of Sirigen Group Ltd.

1. Gerdes J, et al. 1983. Int. J. Cancer 31:13. (IHC)
2. Gerdes J, et al. 1984. J. Immunol. 133:1710. (ICFC)
3. Schluter C, et al. 1993 J. Cell Biol. 123:513. (IHC, WB)
4. Bading H, et al. 1989 Exp. Cell. Res. 185:50. (IF)
5. Guha P, et al. 2013. PNAS. 110:5052. PubMed

Expressed in the phases G₁, S, G₂, and M of the cell cycle

1. Byeon IJ, et al. 2005. Nat. Struct. Mol. Biol. 12:987.
2. Yerushalmi R, et al. 2010. Lancet. Oncol. 11:174.
3. Beltrami AP, et al. 2001. N. Engl. J. Med. 344:1750.
4. Sachsenberg N, et al. 1998. J. Exp. Med. 187:1295.
5. Nagy Z, et al. 1997. Acta. Neuropathol. 93:294.