BD Pharmingen™ Apoptosis, DNA Damage and Cell Proliferation Kit

Kit Reagents

Some kit reagents are supplied as concentrated stock solutions and need to be diluted either with deionized water, 1X Dulbecco's PBS (DPBS), or with BD Perm/Wash Buffer. Instructions for handling, preparation, and storage of kit components is as follows:

PerCP-Cy5.5 Mouse Anti-BrdU Antibody. This vial contains 250 μl of the stock solution of PerCP-Cy5.5 Anti-BrdU antibody that is sufficient for staining 50 samples (10^6 cells/sample). This fluorescent antibody should be stored in the dark at 4°C. Add 5 μl of the stock antibody solution to the sample.

Alexa Fluor™ 647 Mouse Anti-H2AX (pS139) Antibody. This vial contains 250 μl stock solution of Alexa Fluor™ 647 Anti-H2AX (pS139) antibody that is sufficient for staining 50 samples (10^6 cells/sample). This fluorescent antibody should be stored in the dark at 4°C. Add 5 μl of the stock antibody solution to the sample.

PE Mouse Anti-Cleaved PARP (Asp214) Antibody. This vial contains 250 μl of stock solution of PE Anti-Cleaved PARP (Asp214) conjugated antibody that is sufficient for staining 50 samples (10^6 cells/sample). This fluorescent antibody should be stored in the dark at 4°C. Add 5 μl of the stock antibody solution to the sample.

DAPI (4',6-diamidino-2-phenylindole dihydrochloride) is a fluorescent dye for labeling DNA for flow cytometric analysis. Stock solution is at 1 mg/ml in water. Add 1 μl of the DAPI stock solution per ml of staining buffer. One vial of DAPI is provided and should be stored in the dark at 4ºC.

BD Cytofix/Cytoperm™ Plus Permeabilization Buffer. BD Cytofix/Cytoperm Plus Permeabilization Buffer is specially formulated for the Apoptosis, DNA Damage, and Cell Proliferation Kit and is used as a staining enhancer and secondary permeabilization reagent (100 μl/sample). One 10 ml bottle of BD Cytoperm Plus Buffer is provided and should be stored at 4°C.

Note: The BD Cytoperm Plus Buffer should be used with fixed cell samples only. Use of this buffer with unfixed cells will cause cell damage.

BrdU. Each vial contains 0.5 ml of a 10 mg of BrdU/ml (32.5 mM) solution diluted in 1X DPBS. The BrdU solution is prepared aseptically (0.22 μm filtered) and contains no preservative; therefore it is recommended that the solution be handled under aseptic conditions. This stock solution can be injected intraperitoneally (i.p.) into animals or diluted to a 1 mM solution for in vitro labeling of cells. For in vivo labeling of S-phase cycling cells by i.p. injection, proceed to the in vivo labeling section of the Technical Data Sheet (page 4). To label cells in vitro, first dilute the stock (10 mg of BrdU /ml solution) to a 1 mM solution by adding 31 μl of the BrdU stock solution to either 1 ml of 1X DPBS or culture medium (this is a dilution of 32X). Add 10 μl of the 1 mM BrdU solution to each ml of culture medium to obtain a final concentration of 10 μM. The molecular weight of BrdU is 307.1. Five vials of BrdU Solution are provided and should be stored at -80°C.

Note: The BrdU solution has been shown to be stable for up to 4 months at 4˚C or can be refrozen. Avoid multiple freeze-thaw cycles.

DNase. Each vial contains 300 μl of a 1 mg of DNase/ml solution in 1X DPBS. When staining 10 or more samples, thaw the entire vial of DNase solution and add 700 μl of 1X DPBS to make a working stock solution of 300 μg/ml.

Note: If fewer than 10 samples are being treated with DNase, take a 30 μl aliquot of (1 mg/ml) DNase solution/sample and refreeze the remaining 1 mg/ml DNase at -80°C.

Note: DNase stock solution (ie, at 1 mg DNase/ml) may be refrozen once before it loses activity. A total of 100 μl of the working stock is used to treat each cell sample (ie, 30 μg of DNase/10^6 cells) with incubations performed at 37°C. Five vials of DNase are provided and should be stored at -80°C.

BD Cytofix/Cytoperm™ Fixation/Permeabilization Solution. BD Cytofix/Cytoperm Fixation/Permeabilization Solution constitutes a single step fixation and permeabilization reagent that is designed for use in intracellular staining. It contains a mixture of the fixative paraformaldehyde and the detergent saponin. This reagent serves to preserve cell morphology, fix cellular proteins, and permeabilize cells for the subsequent immunofluorescent staining of intracellular proteins. A 25 ml bottle of BD Cytofix/Cytoperm Fixation/Permeabilization Solution is provided in a ready to use formulation. BD Cytofix/Cytoperm Fixation/Permeabilization Solution should be stored at 4°C.

BD Perm/Wash™ Buffer (10X). Each 25 ml bottle contains a concentrated (10X) stock solution of BD Perm/Wash Buffer. The BD Perm/Wash Buffer mixture contains fetal bovine serum and the reversible permeabilization detergent reagent, saponin. The concentrated stock buffer should be diluted 1:10 with deionized water; unused portions of 1X BD Perm/Wash Buffer may be stored at 4°C. The two bottles of 10X BD Perm/Wash Buffer that are provided should be stored at 4°C.

Note: The presence of some precipitate in the 10X BD Perm/Wash stock buffer is common. The precipitate will not affect the performance of the buffer. If desired, the precipitate may be removed prior to use by filtration of the diluted 1X BD Perm/Wash Buffer through a 0.45 μm-pore filter.

Note: The BD Perm/Wash Buffer (1X) should be used with fixed cell samples only. Use of this buffer on unfixed cells will cause cell damage.

Note: Source of all serum proteins is from USDA inspected abattoirs located in the United States.

Protocols for Labeling Cells with BrdU

In vitro-labeling of cultured cells and cell lines with BrdU

Many different protocols for in vitro BrdU labeling of cells have been reported. We have found that incubating cells with BrdU at a final concentration of 10 μM in cell culture medium (ie, 10 μl of 1 mM BrdU per ml of culture medium) was effective for labeling a wide variety of human and mouse cell lines and normal cell populations. Prolonged exposure of cells to BrdU allows for the identification of actively-cycling cell populations. Pulse labeling of cells by brief BrdU exposures at various time points permits the determination of cell-cycle kinetics.

To label cells in vitro, carefully add 10 μl of BrdU solution (1 mM BrdU in 1X DPBS) directly to each ml of tissue culture media. For this step, it is important to avoid disturbing the cells in any way (eg, by centrifugation steps or temperature changes) that may disrupt their normal cell cycling patterns. The cell culture density should not exceed 2 × 10^6 cells/ml. The treated cells are then incubated for the desired length of time. For pulse-labeling experiments, the choice of time points and lengths of time for pulsing depend on the test cell population's rate of cell cycle entry and progression. For example, an effective length of time for pulsing an actively proliferating cell line (eg, CTLL-2 cells) is 30-45 minutes (ie, when the cells are in the logarithmic phase of cell proliferation). Researchers should determine time points and pulse-labeling time intervals that are optimal for each different cell line or cell population within a particular experimental system. Cells from the same population that are not BrdU-labeled are the recommended negative staining control for this assay. This will allow determination of background staining levels for the anti-BrdU monoclonal antibody.

Methods for in vivo-labeling of mouse cells with BrdU

Two common methods reported for in vivo BrdU labeling of cells include the intraperitoneal (i.p.) injection of a BrdU-containing solution into mice and the feeding of mice with BrdU that is added to their drinking water. Please note however that these methods are not routinely tested at BD Biosciences Pharmingen.

Method No. 1: Method for injecting BrdU via the intraperitoneal route. A 10 mg/ml solution of BrdU in sterile 1X DPBS is provided for in vivo use. Inject mice i.p. with 100-200 μl (1-2 mg) of BrdU solution. Incorporation of BrdU can be readily detected in thymus and bone marrow derived cells in as little as 1 hr post injection.

Method No. 2: Introduction of BrdU through drinking water. Dilute BrdU to 0.8 mg/ml in the drinking water. The BrdU mixture should be made up fresh and changed daily. Prolonged feeding of BrdU can have toxic effects for the animal. Some researchers have reported lethal effects associated with 14 days of continuous BrdU feeding. For longer term studies, some researchers have reported that feeding mice with BrdU for 9 consecutive days followed by a changeover to normal water has worked effectively. BrdU incorporation by cells from these animals has been detected past 70 days.

Apoptosis, DNA Damage and Cell Proliferation Kit Staining Protocol

1. Immunofluorescent staining of cell surface antigens.

a. Add BrdU-pulsed cells (10^6 cells in 50 μl of staining buffer) to flow cytometry tubes.

b. Add fluorescent antibodies specific for cell-surface markers in 50 μl of staining buffer (eg, BD Pharmingen Stain Buffer (FBS) Cat. No. 554656) per tube and mix well.

c. Incubate cells with antibodies for 15 minutes on ice.

d. Wash cells 1X by adding 1 ml of staining buffer per tube, centrifuge (5 min) at 200-300g, and discard supernatant.

2. Fix and permeabilize cells with BD Cytofix/Cytoperm Fixation/Permeabilization Solution.

a. Resuspend cells with 100 μl of BD Cytofix/Cytoperm Fixation/Permeabilization Solution per tube.

b. Incubate cells for 15-30 minutes at room temperature or on ice.

c. Wash cells 1X with 1 ml of 1X BD Perm/Wash Buffer, centrifuge as in step 1d and discard supernatant.

3. Incubate cells with BD Cytofix/Cytoperm Plus Permeabilization Buffer.

a. Resuspend cells with 100 μl of BD Cytofix/Cytoperm Plus Permeabilization Buffer per tube.

b. Incubate cells for 10 minutes on ice.

c. Wash cells 1X by adding 1 ml of 1X BD Perm/Wash Buffer (as in Step 2c).

4. Re-Fixation of cells

a. Resuspend cells with 100 μl of BD Cytofix/Cytoperm Fixation/Permeabilization Solution per tube.

b. Incubate cells for 5 minutes at room temperature or on ice.

c. Wash cells 1X by adding 1 ml of 1X BD Perm/Wash Buffer (as in Step 2c).

5. Treatment of cells with DNase to expose incorporated BrdU.

a. Resuspend cells with 100 μl of diluted DNase (diluted to 300 μg/ml in DPBS) per tube, (ie, 30 μg of DNase to each tube).

b. Incubate cells for 1 hour at 37°C.

c. Wash cells 1X by adding 1 ml of 1X BD Perm/Wash Buffer (as in Step 2c).

6. Stain BrdU, H2AX (P139), Cleaved PARP (Asp214) and intracellular /surface antigens with fluorescent antibodies.

a. Resuspend cells with 20 μl BD Perm/Wash Buffer + PerCP-Cy™5.5 Anti-BrdU (5 μl/test), Alexa Fluor® 647 Mouse Anti-H2AX (pS139) (5 μl/test), PE Anti-Cleaved PARP (Asp214) (5 μl/test) antibodies with or without other antibodies specific for intracellular or surface antigens.

b. Incubate cells for 20 minutes at room temperature.

c. Wash cells 1X by adding 1 ml of 1X BD Perm/Wash Buffer (as in Step 2c).

7. Optional - Staining of total DNA for cell cycle analysis.

a. Resuspend cells with 1 ml of DAPI solution (1 μg/ml).

b. Analyze stained cells with a flow cytometer (run at a rate no greater than 400 events/sec.) and acquire multiparameter data files.

Note: If you perform the optional staining in Step 7, you are done and can proceed to analyze your cell samples. Otherwise, proceed to Step 8 if the staining of total DNA levels is not desired.

Note: Samples may be stored overnight at 4°C, protected from

8. Resuspension of cells for Flow Cytometric Analysis.

a. Add 1 ml of staining buffer to each tube to resuspend cells.

b. Analyze stained cells with a flow cytometer (run at a rate no greater than 400 events/sec.) and acquire multiparameter data files.

Note: Samples may be stored overnight at 4°C, protected from light.

Recommended controls for instrument setup

Each individual antibody is recommended as the control for instrument set up.

1. PerCP-Cy™5.5 Anti-BrdU (Display in Log Mode)

2. PE Anti-Cleaved PARP (Asp214) (Display in Log Mode)

3. Alexa Fluor® 647 Mouse Anti-H2AX (pS139) (Display in Log Mode)

4. DAPI (Display in Linear Mode)

Use the autocompensation in BD FACSDiva™ software to compensate for the experiment. For the DAPI signal, select the entire DNA histogram population. DAPI is displayed in the linear mode and the G0/G1 peak is set to the 50,000 channel on the histogram.

Danger: BD Cytofix/Cytoperm™ Fixation/Permeabilization Solution (component 51-2090KE) contains 4.2% formaldehyde (w/w).

Hazard Statements:

Causes skin irritation.

Causes serious eye damage.

May cause an allergic skin reaction.

Suspected of causing genetic defects.

May cause cancer.

Precautionary statements:

Obtain special instructions before use. Do not handle until all safety precautions have been read and understood.

Wear protective clothing / eye protection.

Wear protective gloves.

Contaminated work clothing should not be allowed out of the workplace. Take off contaminated clothing and wash it before reuse.

Wash thoroughly after handling.

Do not breathe mist/vapours/spray.

IF IN EYES: Rinse cautiously with water for several minutes. Remove contact lenses, if present and easy to do.

Continue rinsing.

If skin irritation or rash occurs: Get medical advice/attention.

IF ON SKIN: Wash with plenty of water.

IF exposed or concerned: Get medical advice/attention.

Avoid release to the environment.

Store locked up.

Dispose of contents/container to an appropriate treatment and disposal facility in accordance with applicable laws and regulations, and product characteristics at time of disposal.

Danger. BrdU (component 51-2420KC) contains 0.98% 5-Bromo-2ʹ-deoxyuridine (w/w).

Hazard statement:

May cause genetic defects. Suspected of damaging fertility or the unborn child.

Precautionary statements:

Obtain special instructions before use. Do not handle until all safety precautions have been read and understood.

Wear protective gloves/protective clothing/eye protection/face protection.

IF exposed or concerned: Get medical advice/attention.

Store locked up

Dispose of contents/container to an appropriate treatment and disposal facility in accordance with applicable laws and regulations, and product characteristics at time of disposal.

1. 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).
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. Please observe the following precautions: Absorption of visible light can significantly alter the energy transfer occurring in any tandem fluorochrome conjugate; therefore, we recommend that special precautions be taken (such as wrapping vials, tubes, or racks in aluminum foil) to prevent exposure of conjugated reagents, including cells stained with those reagents, to room illumination.
5. PerCP-Cy5.5–labelled antibodies can be used with FITC- and R-PE–labelled reagents in single-laser flow cytometers with no significant spectral overlap of PerCP-Cy5.5, FITC, and R-PE fluorescence.
6. PerCP-Cy5.5 is optimized for use with a single argon ion laser emitting 488-nm light. Because of the broad absorption spectrum of the tandem fluorochrome, extra care must be taken when using dual-laser cytometers, which may directly excite both PerCP and Cy5.5™. We recommend the use of cross-beam compensation during data acquisition or software compensation during data analysis.
7. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
8. Before staining with this reagent, please confirm that your flow cytometer is capable of exciting the fluorochrome and discriminating the resulting fluorescence.
9. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
10. This product is provided under an intellectual property license between Life Technologies Corporation and BD Businesses. The purchase of this product conveys to the buyer the non-transferable right to use the purchased amount of the product and components of the product in research conducted by the buyer (whether the buyer is an academic or for-profit entity). The buyer cannot sell or otherwise transfer (a) this product (b) its components or (c) materials made using this product or its components to a third party or otherwise use this product or its components or materials made using this product or its components for Commercial Purposes. Commercial Purposes means any activity by a party for consideration and may include, but is not limited to: (1) use of the product or its components in manufacturing; (2) use of the product or its components to provide a service, information, or data; (3) use of the product or its components for therapeutic, diagnostic or prophylactic purposes; or (4) resale of the product or its components, whether or not such product or its components are resold for use in research. For information on purchasing a license to this product for any other use, contact Life Technologies Corporation, Cell Analysis Business Unit Business Development, 29851 Willow Creek Road, Eugene, OR 97402, USA, Tel: (541) 465-8300. Fax: (541) 335-0504.
11. Please refer to http://regdocs.bd.com to access safety data sheets (SDS).
12. Alexa Fluor™ is a trademark of Life Technologies Corporation.
13. Cy is a trademark of Global Life Sciences Solutions Germany GmbH or an affiliate doing business as Cytiva.