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Alexa Fluor® 647 Mouse anti-BrdU
商品详情
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BD Pharmingen™
5-bromo-2'-deoxyuridine, 5-Bromouracil deoxyriboside, BUdR
Mouse IgG1, κ
Bioimaging (Routinely Tested)
5 µl
AB_1645615
Aqueous buffered solution containing BSA and ≤0.09% sodium azide.
RUO


准备和存储

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 to Alexa Fluor® 647 under optimum conditions, and unreacted Alexa Fluor® 647 was removed.

推荐的实验流程

Cells are pulse labeled with BrdU, fixed to maintain BrdU loading, and permeabilized for intranuclear staining of the incorporated BrdU.  DNase I is used to randomly cleave the DNA, allowing the anti-BrdU monoclonal antibody to bind to the incorporated BrdU.

Materials

        Adherent cell culture growing in a Falcon™ 96-well Imaging Plate

        37°C incubator

        BrdU (Cat. No. 550891) diluted to 10-100 μM in tissue culture medium, prepare enough to use at 50 μl per well (see step 1 of procedure)

        BD Cytofix™ fixation buffer (Cat. No. 554655), warmed to 37°C

        BD™ Phosflow Perm Buffer III (Cat. No. 558050), at -20°C

        Phosphate-buffered saline solution (PBS), at room temperature

        BD Pharmingen™ Stain Buffer (FBS) (Cat. No. 554656), at 4°C

        300 μg/ml sterile solution of DNase I (preferred, Sigma-Aldrich Cat. No. D4513) in PBS, prepare enough to use at 50 μl per well

        Alexa Fluor® 647 Mouse anti-BrdU monoclonal antibody diluted 1:10 in BD Pharmingen™ Stain Buffer (FBS), prepare enough to use at 50 μl per well.  If additional antibodies are to be used, they should be added so that all antibodies are included in the 50 μl per well.

        2 μg/ml solution of Hoechst 33342 (eg, Sigma-Aldrich Cat. No. B2261) in PBS, prepare enough to use at 100 μl per well

        Fluorescence imaging instrument capable of exciting Alexa Fluor® 647 and Hoechst 33342, such as the BD Pathway™ Bioimaging System

Procedure

        1.        Pulse the adherent cell cultures by adding 50 μl of the diluted BrdU to each well and incubating for 1-2 hrs at 37°C.  Omit the BrdU from the wells that will be used as negative controls for the BrdU staining.  In all subsequent steps, the BrdU-loaded and control cells should be processed in parallel.

        2.        Remove the medium from the wells, and fix the cells by adding 100 μl of the pre-warmed fixation buffer to each well and incubating for 10 - 20 minutes at room temperature.

        3.        Remove the fixative from the wells, and wash the wells twice with 100 μl of PBS.

        4.        Remove the PBS, and permeabilize the cells by adding 100 μl of the ice-cold BD™ Phosflow Perm Buffer III to each well and incubating for 5 - 10 minutes at room temperature.

        5.        Remove the Perm Buffer III from the wells, and wash the wells twice with 100 μl of PBS.

        6.        OPTIONAL: Remove the PBS, and block the cells by adding 100 μl of the BD Pharmingen™ Stain Buffer (FBS) to each well and incubating for 15 - 30 minutes at room temperature.

        7.        Remove the BD Pharmingen™ Stain Buffer (FBS), and denature the cells' DNA by adding 50 μl of the sterile DNase I solution to each well and incubating for 1 hour at 37°C.

        8.        Remove the DNase I solution from the wells, and wash the wells once with 100 μl of PBS.

        9.        Remove the PBS, and stain the cells by adding 50 μl of the diluted antibody solution to each well and incubating for 1 hour at room temperature.

      10.        Remove the antibody solution, and wash the wells twice with 100 μl of PBS.

      11.        Remove the PBS, and counter-stain the nuclei by adding 100 μl of the Hoechst 33342 solution to each well at least 15 minutes before imaging.

      12.        View and analyze the cells on an appropriate imaging instrument.  Recommended filters for the BD Pathway™ cell analyzers are:

Instrument                        Excitation        Emission        Dichroic

BD Pathway 855                620/60                700/75                660 LP

BD Pathway 435                628/40                690/40                FF660

商品通知

  1. This reagent has been pre-diluted for use at the recommended Volume per Test when following the Recommended Assay Procedure. A Test is typically ~10,000 cells cultured in a well of a 96-well imaging plate.
  2. Source of all serum proteins is from USDA inspected abattoirs located in the United States.
  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. Alexa Fluor® is a registered trademark of Molecular Probes, Inc., Eugene, OR.
  5. The Alexa Fluor®, Pacific Blue™, and Cascade Blue® dye antibody conjugates in this product are sold under license from Molecular Probes, Inc. for research use only, excluding use in combination with microarrays, or as analyte specific reagents. The Alexa Fluor® dyes (except for Alexa Fluor® 430), Pacific Blue™ dye, and Cascade Blue® dye are covered by pending and issued patents.
  6. Alexa Fluor® 647 fluorochrome emission is collected at the same instrument settings as for allophycocyanin (APC).
  7. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
560209 Rev. 2
抗体详情
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3D4

Bromodeoxyuridine (BrdU) is an analog of thymidine that can be incorporated into newly synthesized DNA by cells entering and progressing through the DNA synthesis (S) phase of the cell cycle.  The amount of incorporated BrdU depends on the amount of time that the cells are exposed to BrdU (pulse time), the rate of cell division, and whether the cells are in early, mid, or late S phase. Investigators can identify cycling cells in an asynchronous cell population and determine cell cycle kinetics by detecting incorporated BrdU.

The 3D4 monoclonal antibody reacts with BrdU, but not other nucleotides, in single-stranded DNA. Random cleavage (nicking) of cellular DNA with DNase I permits the binding of the antibody to incorporated BrdU.

560209 Rev. 2
格式详情
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Alexa Fluor™ 647
Alexa Fluor™ 647 Dye is part of the BD red family of dyes. This is a small organic fluorochrome with an excitation maximum (Ex Max) at 653-nm and an emission maximum (Em Max) at 669-nm. Alexa Fluor 647 is designed to be excited by the Red laser (627-640 nm) and detected using an optical filter centered near 520-nm (e.g., a 660/20 nm bandpass filter). Please ensure that your instrument’s configurations (lasers and optical filters) are appropriate for this dye.
Alexa Fluor™ 647
Red 627-640 nm
653 nm
669 nm
560209 Rev.2
报价单和参考
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研发参考 (1)

  1. Miltenburger HG, Sachse G, Schliermann M. S-phase cell detection with a monoclonal antibody. Dev Biol Stand. 1987; 66:91-99. (Clone-specific: Immunofluorescence).
560209 Rev. 2

Please refer to Support Documents for Quality Certificates


Global - Refer to manufacturer's instructions for use and related User Manuals and Technical data sheets before using this products as described


Comparisons, where applicable, are made against older BD Technology, manual methods or are general performance claims.  Comparisons are not made against non-BD technologies, unless otherwise noted.

For Research Use Only. Not for use in diagnostic or therapeutic procedures.