CytoSelect™ BrdU Cell Proliferation ELISA Kit

Catalog No. ABIN2344922

Product Details

Target: BrdU (Bromodeoxyuridine (BrdU))

Reactivity: Mammalian

Detection Method: Colorimetric

Method Type: Cell ELISA

Application: ELISA, Proliferation Assay (ProA)

Purpose: The CytoSelect™ BrdU Cell Proliferation ELISA Kit detects BrdU incorporated into cellular DNA during cell proliferation using an anti-BrdU antibody.

Brand: CytoSelect™

Sample Type: Cell Samples

Components:

1. 1000X BrdU Solution : One 30 μL vial of 10 mM BrdU.
2. Anti-BrdU Monoclonal Antibody : One 10 μL vial of mouse anti-BrdU antibody.
3. Fix/Denature Solution : One 20 mL bottle.
4. Antibody Diluent : One 50 mL bottle.
5. Secondary Antibody, HRP Conjugate : One 20 μL vial.
6. 10X Wash Buffer : One 100 mL bottle.
7. Substrate Solution : One 12 mL amber bottle.
8. Stop Solution (Part. No. 310808): One 12 mL bottle.

Material not included:

1. Mammalian Cells
2. Cell growth media
3. PBS
4. Deionized water
5. 10 μL to 1000 μL adjustable single channel micropipettes with disposable tips
6. 50 μL to 300 μL adjustable multichannel micropipette with disposable tips
7. 96 well cell culture plate
8. Multichannel micropipette reservoir
9. Microplate reader capable of reading at 450 nm (620 nm as optional reference wave length)

Application Details

Application Notes: Optimal working dilution should be determined by the investigator.

Comment:

• Detects BrdU incorporated into cellular DNA during cell proliferation using an anti-BrdU antibody
• BrdU standard included

Plate: Uncoated

Protocol: When cells are incubated in media containing BrdU, the pyrimidine analog is incorporated in place of thymidine into the newly synthesized DNA of proliferating cells . Once the labeling media is removed, the cells are fixed and the DNA is denatured in one step with a fix/denature solution (denaturation of the DNA is necessary to improve the accessibility of the incorporated BrdU for detection). Then an anti-BrdU mouse monoclonal antibody is added followed by an HRP conjugated secondary antibody to detect the incorporated BrdU. The magnitude of the absorbance for the developed color is proportional to the quantity of BrdU incorporated into cells and can be directly correlated to cell proliferation. : Schematic of the CytoSelect™ BrdU Proliferation ELISA

Reagent Preparation:

• 1X Wash Buffer: Dilute the 10X Wash Buffer to 1X with deionized water. Stir to homogeneity.
• Anti-BrdU Monoclonal Antibody and Secondary Antibody, HRP Conjugate: Immediately before use dilute the Anti-BrdU Monoclonal Antibody 1:1000 with Antibody Diluent. Immediately before use dilute the Secondary Antibody, HRP Conjugate 1:1000 with Antibody Diluent. Do not store diluted solutions.
• 10X BrdU Solution: Immediately before use, dilute 1000X stock of BrdU 1:100 with cell growth media.

Assay Procedure:

1. Prepare a cell suspension containing 0.1-1.0 x 106 cells/mL in medium. 3
2. Add 100 μL per well to a 96-well cell culture plate and incubate overnight at 37 °C and 5 % CO2 in a humidified incubator.
3. Add compound to be tested and include wells without compound (or with vehicle) as a negative control. Culture the cells for 24-96 hours at 37 °C and 5 % CO2 in a humidified incubator.
4. Add 10 μL of 10X BrdU Solution (see Preparation of Reagents Section) to wells and incubate at 37 °C and 5 % CO2 in a humidified incubator for 1-6 hours. Note: optimal time of incubation with BrdU will vary with cell type.
5. Carefully and slowly aspirate wells by pipette and add 100 μL PBS, repeat this wash step 2 more times.
6. After the final aspiration, add 100 μL Fix/Denature Solution and incubate 30 minutes at 37 °C.
7. Wash wells 3 times 100 μL per well with PBS as described in step
8. 8. Add 100 μL Antibody Diluent and incubate 1 hour at room temperature.
9. Wash wells 3 times with 100 μL PBS.
10. Add 100 μL of diluted Anti-BrdU Antibody (see Preparation of Reagents section) to each tested well. Incubate at room temperature for 1 hour on an orbital shaker.
11. Wash wells 3 times with 250 μL 1X Wash Buffer per well as described above for PBS washes.
12. Add 100 μL of the diluted Secondary Antibody HRP Conjugate (see Preparation of Reagents section) to each well. Incubate at room temperature for 1 hour on an orbital shaker. During this incubation, warm Substrate Solution to room temperature.
13. Wash the strip wells 3 times according to step
14. Proceed immediately to the next step.
15. Add 100 μL of Substrate Solution to each well, including the blank wells. Incubate at room temperature on an orbital shaker. Actual incubation time may vary from 2-30 minutes. Note: Watch plate carefully, if color changes rapidly, the reaction may need to be stopped sooner to prevent saturation.
16. Stop the enzyme reaction by adding 100 μL of Stop Solution into each well, including the blank wells. Results should be read immediately (color will fade over time).
17. Read absorbance of each microwell on a spectrophotometer using 450 nm as the primary wave length. 4

Restrictions: For Research Use only

Handling

Storage: 4 °C/-20 °C

Storage Comment: Upon receipt, store the 1000X BrdU Solution and Anti-BrdU Monoclonal Antibody at -20°C. Store all other components at 4°C.

References

Kim, Tilstam, Hwang, Simons, Schulte, Leng, Sauler, Ganse, Averdunk, Kopp, Stoppe, Bernhagen, Pallua, Bucala: "D-dopachrome tautomerase in adipose tissue inflammation and wound repair." in: Journal of cellular and molecular medicine, (2016) (PubMed).

Kreiseder, Holper-Schichl, Muellauer, Jacobi, Pretsch, Schmid, de Martin, Hundsberger, Eger, Wiesner: "Alpha-catulin contributes to drug-resistance of melanoma by activating NF-?B and AP-1." in: PLoS ONE, Vol. 10, Issue 3, pp. e0119402, (2015) (PubMed).

Hatzis, Bedard, Birkbak, Beck, Aerts, Stem, Stern, Shi, Clarke, Quackenbush, Haibe-Kains: "Enhancing reproducibility in cancer drug screening: how do we move forward?" in: Cancer research, Vol. 74, Issue 15, pp. 4016-23, (2014) (PubMed).

Target Details

Target: BrdU (Bromodeoxyuridine (BrdU))

Alternative Name: BrdU (BrdU Products)

Target Type: Chemical

Background: The investigation of cell cycle and DNA synthesis has been essential to many fields of science. Traditionally a radiolabled thymidine has been used to track new DNA synthesis and cellular proliferation. Although quite sensitive, use of radiolabled thymidine has the limitation of having to regulate, handle and dispose of radioisotopes and often requires expensive detection equipment. More recently, the thymidine analog 5-bromo-2'-deoxyuridine (BrdU) has been used in place of radiolabled thymidine and is incorporated into newly synthesized DNA strands of actively proliferating cells. Following fixation and partial denaturation of cellular DNA, BrdU can be detected immunochemically which allows for the analysis of live cell new DNA synthesis.