Description
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.
STAINING PROTOCOL FOR 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 votexing, add 5 ml drop by drop of cold 70-80% ethanol into the cells pellet (1-5x10^7 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, pH 7.2) to the fixed cells. Centrifuge the cells for 10 minutes at 1000 rpm and aspirate supernatant. Wash one more time with 30-40 ml wash buffer. Centrifuge at 1000 rpm for 10 minutes and aspirate supernatant. 4. Resuspend the cells to a concentration of 1 x 10^7/ml (1 x 10^6/100 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 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. STAINING PROTOCOL FOR BIOIMAGING: Methanol Procedure for a 96 well plate: Remove media from wells. Add 100 µl/well fresh 3.7% Formaldehyde in PBS. Incubate for 10 minutes at room temperature (RT). Flick out and add 100 µl/well 90% methanol. Incubate for 5 minutes at RT. Flick out and wash twice with PBS. Flick out PBS and add 100 µl/well blocking buffer (3% FBS in PBS). Incubate for 30 minutes at RT. Flick out and add diluted antibody (diluted in blocking buffer). Incubate for 1 hour at RT. Wash three times with PBS. Flick out PBS and add second step reagent. Incubate for 1 hour at RT. Wash three times with PBS. Image sample. Triton-X 100 Procedure for a 96 well plate: Remove media from wells. Add 100 µl/well fresh 3.7% Formaldehyde in PBS. Incubate for 10 minutes at room temperature (RT). Flick out and add 100 µl/well 0.1% Triton™ X-100. Incubate for 5 minutes at RT. Flick out and wash twice with PBS. Flick out PBS and add 100 µl/well blocking buffer (3% FBS in PBS). Incubate for 30 minutes at RT. Flick out and add diluted antibody (diluted in blocking buffer). Incubate for 1 hour at RT. Flick out and wash three times with PBS. Flick out and add second step reagent. Incubate for 1 hour at RT. Flick out and wash three times with PBS. Image sample.
Store undiluted at 4°C. The monoclonal antibody was purified from tissue culture supernatant or ascites by affinity chromatography.
1. Since applications vary, each investigator should titrate the reagent to obtain optimal results.
2. An isotype control should be used at the same concentration as the antibody of interest.
3. 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.
4. This antibody has been developed and certified for the bioimaging application. However, a routine bioimaging test is not performed on every lot. Researchers are encouraged to titrate the reagent for optimal performance.
5. Species cross-reactivity detected in product development may not have been confirmed on every format and/or application.
6. Sodium azide is a reversible inhibitor of oxidative metabolism; therefore, antibody preparations containing this preservative agent must not be used in cell cultures nor injected into animals. Sodium azide may be removed by washing stained cells or plate-bound antibody or dialyzing soluble antibody in sodium azide-free buffer. Since endotoxin may also affect the results of functional studies, we recommend the NA/LE (No Azide/Low Endotoxin) antibody format, if available, for in vitro and in vivo use.
7. Triton is a trademark of the Dow Chemical Company.
8. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
