BV650 Mouse Anti-Ki-67
Clone B56 (RUO)
- Brand BD Horizon™
- Alternative Name MKI67; Antigen identified by monoclonal antibody Ki-67; KIA
- Vol. Per Test 5 µl
- Isotype Mouse IgG1, κ
- Reactivity Human (QC Testing) Mouse (Tested in Development) Rat, Rhesus (Reported)
Intracellular staining (flow cytometry) (Routinely Tested)
- Immunogen Human Ki-67
- Storage Buffer Aqueous buffered solution containing BSA and ≤0.09% sodium azide.
- Regulatory Status RUO
Regulatory Status Legend
The B56 monoclonal antibody specifically binds to the Ki-67 antigen that is expressed in the nucleus of cycling cells (G1, S, G2, M cell cycle phases). During the G0 phase, the antigen cannot be detected. During interphase of the cell cycle, it is associated with nucleolar components, and it is on the surface of the chromosomes during M phase. Ki-67 is a large protein having 2 alternatively spliced isoforms, an N-terminal forkhead-associated domain, a C-terminal domain that binds to heterochromatin proteins, and multiple phosphorylation sites, the functions of which are still unclear. Because of the strict association of Ki-67 expression with cell proliferation, anti-Ki-67 antibodies are useful for the identification, quantification, and monitoring of growing cell populations.
The antibody was conjugated to BD Horizon BV650 which is part of the BD Horizon Brilliant™ Violet family of dyes. This dye is a tandem fluorochrome of BD Horizon BV421 with an Ex Max of 405-nm and an acceptor dye with an Em Max at 650-nm. BD Horizon BV650 can be excited by the violet laser and detected in a filter used to detect APC-like dyes (eg, 660/20-nm filter). Due to the excitation and emission characteristics of the acceptor dye, there will be spillover into the APC and Alexa Fluor® 700 detectors. However, the spillover can be corrected through compensation as with any other dye combination.
BV650 is a tandem fluorochrome of BD Horizon BV421 and an acceptor dye with an emission maximum at 650 nm. Due to the excitation and emission characteristics of the acceptor dye, there will be spillover into the APC and Alexa Fluor® 700 detectors. BV650 will have moderate spillover into the BD Horizon™ BV711 detector.
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Preparation and Storage
Store undiluted at 4°C and protected from prolonged exposure to light. Do not freeze.
The monoclonal antibody was purified from tissue culture supernatant or ascites by affinity chromatography.
The antibody was conjugated with BD Horizon™ BV650 under optimum conditions, and unconjugated antibody and free BD Horizon™ BV650 were removed.
- This reagent has been pre-diluted for use at the recommended Volume per Test. We typically use 1 × 10^6 cells in a 100-µl experimental sample (a test).
- An isotype control should be used at the same concentration as the antibody of interest.
- 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.
- Species cross-reactivity detected in product development may not have been confirmed on every format and/or application.
- Alexa Fluor® is a registered trademark of Molecular Probes, Inc., Eugene, OR.
- BD Horizon Brilliant Violet 650 is covered by one or more of the following US patents: 8,110,673; 8,158,444; 8,227,187; 8,455,613; 8,575,303; 8,354,239.
- Source of all serum proteins is from USDA inspected abattoirs located in the United States.
- For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
- Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
The following protocol encourages using DAPI; higher background or signal spillover may be experienced with 7-AAD or propidium iodide (PI).
1. Harvest, count and pellet cells following standard procedures. Note: Ki-67 is expressed by proliferating cells. Using resting cells may give
2. While vortexing, add 5 mL cold 70% ethanol dropwise into the cell pellet (1-5 x 10^7 cells).
3. Wash twice with 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 10^7 cells/mL.
5. Transfer 100 µL (1 x 10^6 cells) cell suspension into each sample tube.
6. Add 5 µL of BV650 Mouse Anti-Ki-67 antibody into the appropriates tubes. Mix gently.
7. Incubate the tubes at 4°C for 60 min in the dark.
8. Wash with 2 mL of staining buffer at 200 x g for 5 minutes.
9. Aspirate the supernatant.
10. Repeat wash with 2 mL of staining buffer at 200 x g for 5 minutes.
11. Aspirate the supernatant.
12. Resuspend in 350-500 µL DAPI (Cat. No. 564907) diluted to 1 µg/mL.
13. Proceed to flow cytometric analysis.
For optimal and reproducible results, BD Horizon Brilliant Stain Buffer should be used anytime two or more BD Horizon Brilliant dyes are used in the same experiment. Fluorescent dye interactions may cause staining artifacts which may affect data interpretation. The Brilliant Stain Buffer was designed to minimize these interactions. More information can be found in the TDS of the Brilliant Stain Buffer (Cat. No. 563794).