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CBA040 Apo-BrdU™ Kit

Apo-BrdU™ Kit MSDS (material safety data sheet) or SDS, CoA and CoQ, dossiers, brochures and other available documents.
CBA040
  
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Overview

Replacement Information

Key Spec Table

Detection Methods
Fluorescence
Description
Overview

This product has been discontinued.





A two color TUNEL (Terminal deoxynucleotide transferase dUTP Nick End Labeling) assay for labeling DNA breaks and total cellular DNA to detect apoptotic cells by flow cytometry and microscopy. This kit labels the 3'-hydroxy termini of apoptotic DNA fragments with bromolated deoxyuridine triphosphate nucleotides (Br-dUTP). It is known that Br-dUTP is more readily incorporated into the genome of apoptotic cells compared to larger ligands, such as fluorescein or biotin.
Catalogue NumberCBA040
Brand Family Calbiochem®
Materials Required but Not Delivered• Flow cytometer
• Distilled water
• 1% (w/v) paraformaldehyde (methanol free) in phosphate buffered saline (PBS)
• 70% (v/v) ethanol
• 37°C water bath
• Ice bucket
• 12 x 75 mm flow cytometry test tubes
• Pipets and pipetting aids
References
ReferencesLi, X. and Z. Darzynkiewicz, 1995. Cell Prolif. 28, 572.
Li, X., Traganos, F., et al. 1995. Cytometry 20, 172.
Nagata, S., and Golstein, P. 1995. Science 267, 1449.
Steller, H. 1995. Science 26, 1445.
Thompson, C. B. 1995. Science 267, 1456.
Darzynkiewicz, Z., et al. 1994. in Methods in Cell Biology: Flow Cytometry, 2nd edition., Darzynkiewicz, Z., Crissman, H. A., and Robinson, J. R., eds., Academic Press.
Darzynkiewicz, Z., et al. 1992. Cytometry 13, 795.
Lockshin, R.A. and Zakeri, Z. Programmed cell death and apoptosis. In: L. D. Tomei and F. D. Cope (eds.), Current Communications in Cell and Molecular Biology, Vol. 3., pp. 47-60. Cold Springs Harbor, NY: Cold Springs Laboratory, 1991.
Strasser, A., et al. 1991. Cell 67, 889.
Arends, M. J., et al. 1990. Am. J. Pathol. 136, 593.
Hasbold, J., and Klaus, G. G. B. 1990. Eur. J. Immunol. 20, 1685
Benedetti, A., et al. 1988. J. Hepatol. 7, 319.
Buttyan, R., et al. 1988. Mol. Endocrinol. 2, 650.
Eschenfeldt, W. H., et al. 1987. in Enzymology 152: 337-342, Berger, S. L., and Kimmel, A. R., eds., Academic Press, 1987.
Goldman, A. S., et al. 1983. Proc. Soc. Exp. Biol. Med. 174, 239.
Weedon, D., and Strutton, G. 1981. Acta Derm. Venerol. 61, 335.
Appleby, D. W., and Modak, S. P., 1977. Proc. Natl. Acad. Sci. USA 74, 5579.
Product Information
positive and negative control cells, wash buffer, reaction buffer, TdT enzyme, Br-dUTP, rinsing buffer, fluorescein PRB-1 mAb, PI/RNase staining buffer
Detection methodFluorescence
Form60 Tests
FormatFlow cytometry
Kit containsPositive Control Cells, Negative Control Cells, Wash Buffer, Reaction Buffer, TdT, Br-dUTP, Rinsing Buffer, Fluorescein PRB-1 mAb, PI/RNase Staining Buffer, and a user protocol.
Applications
Key Applications Flow Cytometry
Immunofluorescence
Biological Information
Assay time4-5 h
Sample TypeCells
Physicochemical Information
Dimensions
Materials Information
Toxicological Information
Safety Information according to GHS
Safety Information
Product Usage Statements
Intended useThe Calbiochem® Apo-BrdU™ Kit is a two color TUNEL (Terminal deoxynucleotidyl transferase dUTP Nick End Labeling) assay for labeling DNA breaks and total cellular DNA to assess apoptotic cells by flow cytometry and microscopy.
Storage and Shipping Information
Ship Code Blue Ice Only
Toxicity Multiple Toxicity Values, refer to MSDS
Hazardous Materials Attention: Due to the nature of the Hazardous Materials in this shipment, additional shipping charges may be applied to your order. Certain sizes may be exempt from the additional hazardous materials shipping charges. Please contact your local sales office for more information regarding these charges.
Storage Multiple storage requirements
Storage ConditionsThe ApoBrdU™ Kit is shipped in two parts:

The following components are shipped on blue ice and should be stored at -20°C immediately upon arrival:

• Positive Control Cells
• Negative control Cells
• TdT Enzyme
• Br-dUTP

The following components are shipped at ambient temperature and should be stored at 4°C immediately upon arrival:

• Wash Buffer
• Reaction Buffer
• Rinsing Buffer
• Fluorescein~PRB-1 mAb
• PI/Rnase Staining Buffer
Avoid freeze/thaw Avoid freeze/thaw
Do not freeze Ok to freeze
Packaging Information
Transport Information
Supplemental Information
Kit containsPositive Control Cells, Negative Control Cells, Wash Buffer, Reaction Buffer, TdT, Br-dUTP, Rinsing Buffer, Fluorescein PRB-1 mAb, PI/RNase Staining Buffer, and a user protocol.
Specifications
Global Trade Item Number
Catalogue Number GTIN
CBA040 0

Documentation

Apo-BrdU™ Kit Certificates of Analysis

TitleLot Number
CBA040

References

Reference overview
Li, X. and Z. Darzynkiewicz, 1995. Cell Prolif. 28, 572.
Li, X., Traganos, F., et al. 1995. Cytometry 20, 172.
Nagata, S., and Golstein, P. 1995. Science 267, 1449.
Steller, H. 1995. Science 26, 1445.
Thompson, C. B. 1995. Science 267, 1456.
Darzynkiewicz, Z., et al. 1994. in Methods in Cell Biology: Flow Cytometry, 2nd edition., Darzynkiewicz, Z., Crissman, H. A., and Robinson, J. R., eds., Academic Press.
Darzynkiewicz, Z., et al. 1992. Cytometry 13, 795.
Lockshin, R.A. and Zakeri, Z. Programmed cell death and apoptosis. In: L. D. Tomei and F. D. Cope (eds.), Current Communications in Cell and Molecular Biology, Vol. 3., pp. 47-60. Cold Springs Harbor, NY: Cold Springs Laboratory, 1991.
Strasser, A., et al. 1991. Cell 67, 889.
Arends, M. J., et al. 1990. Am. J. Pathol. 136, 593.
Hasbold, J., and Klaus, G. G. B. 1990. Eur. J. Immunol. 20, 1685
Benedetti, A., et al. 1988. J. Hepatol. 7, 319.
Buttyan, R., et al. 1988. Mol. Endocrinol. 2, 650.
Eschenfeldt, W. H., et al. 1987. in Enzymology 152: 337-342, Berger, S. L., and Kimmel, A. R., eds., Academic Press, 1987.
Goldman, A. S., et al. 1983. Proc. Soc. Exp. Biol. Med. 174, 239.
Weedon, D., and Strutton, G. 1981. Acta Derm. Venerol. 61, 335.
Appleby, D. W., and Modak, S. P., 1977. Proc. Natl. Acad. Sci. USA 74, 5579.
User Protocol

Revision02-June-2008 JSW
Form60 Tests
FormatFlow cytometry
Detection methodFluorescence
StorageThe ApoBrdU™ Kit is shipped in two parts:

The following components are shipped on blue ice and should be stored at -20°C immediately upon arrival:

• Positive Control Cells
• Negative control Cells
• TdT Enzyme
• Br-dUTP

The following components are shipped at ambient temperature and should be stored at 4°C immediately upon arrival:

• Wash Buffer
• Reaction Buffer
• Rinsing Buffer
• Fluorescein~PRB-1 mAb
• PI/Rnase Staining Buffer
Intended useThe Calbiochem® Apo-BrdU™ Kit is a two color TUNEL (Terminal deoxynucleotidyl transferase dUTP Nick End Labeling) assay for labeling DNA breaks and total cellular DNA to assess apoptotic cells by flow cytometry and microscopy.
BackgroundApoptosis is the term that describes regulated cell death. It is believed to take place in the majority of animal cells. It is a distinct event that triggers characteristic morphological and biological changes in the cellular life cycle. It is common during embryogenesis, normal tissue and organ involution, cytotoxic immunological reactions and occurs naturally at the end of the life span of differentiated cells. It can also be induced in cells by the application of a number of different agents including physiological activators, heat shock, bacterial toxins, oncogenes, chemotherapeutic drugs, ultraviolet and gamma radiation. When apoptosis occurs, the nucleus and cytoplasm of the cell often fragments into membrane-bound apoptotic bodies that are then phagocytized by neighboring cells. An alternative mode of cell death, necrosis, occurs as a result of gross injury to cells resulting in cellular lysing and release of cytoplasmic components into the surrounding environment, often inducing an inflammatory response in the tissue. A landmark of cellular self destruction by apoptosis is the activation of nucleases that degrade the higher order chromatin structure of the DNA into fragments of 50 to 300 kilobases and subsequently into smaller DNA pieces of about 200 base pairs in length. Numerous reviews of the events accompanying apoptosis are available and several well researched model systems have been described. One of the most easily measured features of apoptotic cells is the breakup of the genomic DNA by cellular nucleases. These DNA fragments can be extracted from apoptotic cells and result in the appearance of "DNA laddering" when the DNA is analyzed by agarose gel electrophoresis. The DNA of non-apoptotic cells, which remains largely intact, does not display this "laddering" on agarose gels during electrophoresis. The large number of DNA fragments appearing in apoptotic cells results in a multitude of 3'-hydroxyl ends in the DNA. This property can be used to identify apoptotic cells by labeling the 3'-hydroxyl ends with bromolated deoxyuridine triphosphate nucleotides (Br-dUTP). The enzyme terminal deoxynucleotidyl transferase (TdT) catalyzes a template independent addition of deoxyribonucleoside triphosphates to the 3'-hydroxyl ends of double- or singlestranded DNA with either blunt, recessed or overhanging ends. A substantial number of these sites are available in apoptotic cells providing the basis for the method utilized in the ApoBrdU™ Kit. Recent evidence has demonstrated that Br-dUTP is more readily incorporated into the genome of apoptotic cells than are the deoxynucleotide triphosphates complexed to larger ligands like fluorescein, biotin or digoxigenin. This greater incorporation gives rise to a stronger flow cytometry signal when the Br-dUTP sites are identified by a fluorescein labeled anti-BrdU monoclonal antibody. Non-apoptotic cells do not incorporate significant amounts of the Br-dUTP owing to the lack of exposed 3'-hydroxyl DNA ends.
Principles of the assay

Figure 1: Principle of the Assay

Diagrammatic representation of the addition of bromodeoxyuridine triphosphate (Br-dUTP) catalyzed by terminal deoxynucleotidyl transferase (TdT) to the 3'-OH sites of DNA strand breaks induced in the genome of apoptotic cells.


The Calbiochem® Apo-BrdU™ Kit is a two color TUNEL (Terminal deoxynucleotide transferase dUTP Nick End Labeling) assay for labeling DNA breaks and total cellular DNA to detect apoptotic cells by flow cytometry and microscopy. The kit contains the instructions and reagents required for measuring apoptosis in cells including positive and negative control cells for assessing reagent performance; washing, reaction, and rinsing buffers for processing individual steps in the assay; terminal deoxynucleotidyl tranferase enzyme (TdT), bromodeoxyuridine triphosphate (Br-dUTP), and fluorescein labeled anti-BrdU antibody for labeling DNA breaks and propidium iodide/RNase A solution for counterstaining the total DNA.
Materials providedReagent bottles have color coded caps to aid in their identification. Sufficient reagents are provided to process 60 cell suspensions including 5 ml positive and 5 ml negative control cell suspensions of approximately 1 x 10⁶ cells per ml in 70% (v/v) ethanol. The control cells are derived from a human lymphoma cell line and have been fixed as described on page 4.

• Positive Control Cells (Kit Component No. JA9101): 1 bottle, 5 ml (brown cap)
• Negative Control Cells (Kit Component No. JA9102): 1 bottle, 5 ml (white cap)
• Wash Buffer (Kit Component No. JA9103): 1 bottle, 120 ml (blue cap)
• Reaction Buffer (Kit Component No. JA9104): 1 vial, 600 µl (green cap)
• TdT Enzyme (Kit Component No. JA9105): 1 vial, 45 µl (yellow cap)
• Br-dUTP (Kit Component No. JA9106): 1 vial, 480 µl (violet cap)
• Rinsing Buffer (Kit Component No. JA9107): 1 bottle, 120 ml (red cap)
• Fluorescein PRB-1 mAb (Kit Component No. JA9108): 1 vial, 300 µl (orange cap)
• PI/RNase Staining Buffer (Kit Component No. JA9109): 1 bottle, 30 ml (amber bottle)
Materials Required but not provided• Flow cytometer
• Distilled water
• 1% (w/v) paraformaldehyde (methanol free) in phosphate buffered saline (PBS)
• 70% (v/v) ethanol
• 37°C water bath
• Ice bucket
• 12 x 75 mm flow cytometry test tubes
• Pipets and pipetting aids
Precautions and recommendations• Component part numbers JA9101 and JA9102 contain 70% (v/v) ethanol as a preservative; JA9104 contains cacodylic acid (dimethylarsenic) as a buffer; JA9103, JA9107, and JA9109 contain 0.05% (w/v) sodium azide as a preservative. These materials are harmful if swallowed; avoid skin contact, wash immediately with water. See Material Safety Data Sheets.
• TdT Enzyme (JA9105) will not freeze at -20°C, because it is in a 50% (v/v) glycerol solution. Upon warming the TdT enzyme solution, centrifuge the tube for 30 s to force all the liquid to the bottom of the tube.
• For those researchers using adherent cell line systems, the cells in the supernatant have a higher probability of being apoptotic than do the adherent cells. Save cells in the supernatant for assay prior to trypsinization of the adherent cell layer.
• Cell fixation using a DNA cross linking chemical fixative is an important step in analyzing apoptosis. Unfixed cells may lose smaller fragments of DNA that are not chemically fixed in place inside the cell during washing steps. The researcher may have to explore alternative fixation and permeablization methods to fully exploit their systems.
• A cytospin or centrifigal cytology slide can be prepared from Apo-BrdU™ Kit sample in the following manner. After completion of the Fluorescein~PRB-1 antibody staining , but prior to the PI/RNAse A treatment, put a drop of the stained cells on a slide, spin it and observe the sample under a fluorescence microscope.
• Surface marker staining of cellular antigens can be accomplished by first incubating the cells with the fluorescent labeled antibody and then fixing and permeabilizing the cells in preparation for the Apo-BrdU™ Kit.
• To minimize cell loss during the assay, restrict the assay to the use of a single 12 X 75 mm test tube. If polystyrene plastic test tubes are used an electrostatic charge can build up on the sides of the tube. Cells will adhere to the side of the tube and the sequential use of multiple tubes can result in significant cell loss during the assay.
• Occasionally a mirror image population of cells at lower intensity is observed in the flow cytometry dual parameter display. This population arises because during the 50 µl DNA Labeling Reaction some cells have become stuck to the side of the test tube and are not fully exposed to the reaction solution. This phenomenon can be overcome by washing all the cells from side of the tube and making sure all cells are properly suspended at the beginning of the labeling reaction.
• If a low intensity of fluorescein staining is observed, try increasing the incubation time during the 50 µl DNA Labeling Reaction. Some researchers have found labeling times of up to 4 h at 37°C may be required for certain cell systems.
• If the DNA cell cycle information is not required, it is not necessary to add the PI/RNase A Staining Solution to each tube.
PreparationCell Fixation Procedure NOTE: Cell fixation using paraformaldehyde is a required step in the Apo-BrdU™ Kit to cross link the DNA in the cells. Ethanol treatment is required to permeabilize the cells. The following cell fixation procedure is a suggested method. Variables such as cell origin and growth conditions can affect the results. The fixation conditions provided below should be considered as guidelines. Additional optimization may be required to obtain results comparable to the control cells provide with this kit. The Positive and Negative Control Cells provided in the Apo-BrdU™ Kit are already fixed as outlined below. 1. Suspend the cells in 1% (w/v) paraformaldehyde in PBS, pH 7.4 at a concentration of 1-2 x 106 cells/ml. 2. Place the cell suspension on ice for 30-60 min. 3. Centrifuge cells for 5 min at 300 x g and discard the supernatant. 4. Wash the cells in 5 ml PBS then pellet the cells by centrifugation. Repeat the wash and centrifugation. 5. Resuspend the cell pellet in the residual PBS in the tube by gently vortexing the tube. 6. Adjust the cell concentration to 1-2 x 106 cells/ml in 70% (v/v) ice cold ethanol. Let cells stand for a minimum of 30 min on ice or in the freezer. See note below. 7. Store cells in 70% (v/v) ethanol at -20°C until use. Cells can be stored at -20°C several days before use. Note: In some biological systems storage of the cells at -20°C in 70% (v/v) ethanol for at least 12-18 h prior to staining for apoptosis detection yields the best results.
Detailed protocolA. Flow Diagram of Apo-BrdU™ Detailed Protocol

Figure 2: Flow Diagram of Apo-BrdU™ Detailed Protocol

Flow diagram used in the Apo-BrdU™ Kit. The Positive and Negative Control Cells are supplied in the kit and are already fixed. The cells supplied by the researcher should be fixed by the researcher according to a protocol suggested in section Detailed Protocol.


B. Protocol
The following protocol describes the method for measuring apoptosis in the Positive and Negative Control cells that are provided in the Apo-BrdU™ Kit. The same procedure should be employed for measuring apoptosis in the cell specimens provided by the researcher.

1. Resuspend the Positive (brown cap) and Negative (natural cap) control cells by swirling the vials. Remove 1 ml aliquots of the control cell suspensions (~1 x 106 cells/ml) and place in 12 x 75 mm flow cytometry centrifuge tubes. Centrifuge (300 x g) the control cell suspensions for 5 min and remove the 70% (v/v) ethanol by aspiration being careful to not disturb the cell pellet.
2. Resuspend each tube of control cells with 1 ml Wash Buffer (blue cap). Centrifuge as before and remove the supernatant by aspiration.
3. Repeat the Wash Buffer treatment (step 2).
4. Resuspend each tube of the control cell pellets in 50 µl DNA Labeling Solution (prepared as described below).

Table 1: DNA Labeling Solution


Note: The appropriate volume of DNA Labeling Solution to prepare for a variable number of assays is based upon multiples of the component volumes combined for 1 Assay. Prepare only enough DNA Labeling Solution to complete the desired number of assays. The DNA Labeling Solution is stable for ~24 h.

5. Incubate the cells in the DNA Labeling Solution for 60 min at 37°C in a temperature controlled bath. Shake cells every 15 min to resuspend.

NOTE: The DNA Labeling Reaction can also be carried out at 22-24°C overnight for the control cells. For samples other than the control cells provided in the kit, incubation times at 37°C may need to be adjusted to longer or shorter periods depending on the characteristics of the cells supplied by the researcher.

6. At the end of the incubation time add 1 ml Rinse Buffer (red cap) to each tube and centrifuge each tube (300 x g) for 5 min. Remove the supernatant by aspiration.
7. Repeat the cell rinsing (as in step 6) with 1 ml Rinse Buffer (red cap), centrifuge and remove the supernatant by aspiration.
8. Resuspend the cell pellets in 100 µl Antibody Solution (prepared as described below).

Table 2: Antibody Solution


9. Incubate the cells with the Antibody Solution in the dark for 30 min at room temperature. Hint: Wrap tubes with aluminum foil.
10. Add 500 µl PI/RNase A Staining Buffer (amber bottle) to each tube.

Note: If the cell density is low, decrease the amount of PI/RNase A solution to 300 µl.

11. Incubate the cells in the dark for 30 min at room temperature.
12. Analyze the cells in PI/RNase A Staining Buffer by flow cytometry.
13. Analyze the cells within 3 h of staining.

C. Analyzing Samples Using a Flow Cytometer

This assay is run on a flow cytometer equipped with a 488 nm Argon laser as the light source. Propidium Iodide (total cellular DNA) and Fluorescein (Apoptotic Cells) are the two dyes being used. Propidium Iodide (PI) fluoresces at ~623 nm and Fluorescein at 520 nm when excited at 488 nm. No fluorescence compensation is required. Two dual parameter and two single parameter displays are created with the flow cytometer data acquisition software. The gating display should be the standard dual parameter DNA doublet discrimination display with the DNA Area signal on the Y-axis and the DNA Width (Becton-Dickinson), see Figure 4 or DNA Peak/Integral (Coulter) signal on the X-axis, see Figure 5. From this display, a gate is drawn around the non-clumped cells and the second gated dual parameter display is generated. The normal convention of this display is to put DNA (Linear Red Fluorescence) on the X-axis and the FITC~PRB-1 (Log Green Fluorescence) on the Y-axis (see bottom display next page). Two single parameter gated histograms, DNA and FITC~PRB-1, can also be added but are not necessary. By using the dual parameter display method, not only are apoptotic cells resolved but at which stage of the cell cycle they are in is also determined. The Log Green Fluorescence histograms of the control cells should look similar to Figure 3 below.
Assay characteristics and examplesA. Flow Cytometry Data for Negative and Positive Control Cells

Figure 3: Flow Cytometry Data for Negative and Positive Control Cells

Flow Cytometry Data using the Apo-BrdU™ Negative & Positive Control Cells.


B. Flow Cytometer Setup for Becton Dickinson Hardware

Figure 4: Flow Cytometer Setup for Becton Dickinson Hardware

Apo-BrdU™ Positive Control Cells.


Figure 5: Flow Cytometer Setup for Coulter Hardware

Apo-BrdU™ Positive Control Cells.
Registered TrademarksCalbiochem® is a registered trademark of EMD Chemicals, Inc.
Interactive Pathways™ is a trademark of EMD Chemicals, Inc.
Apo-BrdU™ is a trademark of Phoenix Flow Systems.