- Apoptosis Kits & Sets
- Buffers and Ancillary Products
- Cell Cycle and Proliferation Kits & Sets
- Conjugated Antibodies
- Purified Antibodies
- Substrates & Inhibitors
In addition, BD Biosciences provides a variety of tools to assist customers in their experimental setup and analysis.
Cell Cycle and Cell Proliferation: An Overview
To help researchers better understand the fundamental cellular mechanisms involved in immunity, inflammation, hematopoiesis, neoplasia, and other biological responses, BD Biosciences offers a range of tools including antibodies, kits, and systems to measure proliferative responses.
Using flow cytometry, immunofluorescence, or immunohistochemistry, researchers can quickly and accurately determine the cell cycle status or tissue localization of individual cells within proliferating populations. These tools include:
- BD Biosciences reagents and BD Cycletest™ Plus reagent kit for the analysis of cellular DNA content
- DNA dyes, propidium iodide (PI), 7-aminoactinomycin D (7-AAD)
- Antibodies against cyclins, retinoblastoma, and phosphorylated histone H3
In adaptive immunity, specific T and B lymphocytes undergo clonal expansion (division, proliferation, and differentiation) in response to foreign antigenic stimulation. Cell growth, replication, and division in eukaryotic cells occur according to a highly controlled series of events called the cell cycle.
The Cell Cycle
The cell cycle has two major phases: interphase, the phase between mitotic events, and the mitotic phase, where the mother cell divides into two genetically identical daughter cells. Interphase has three distinct, successive stages. During the first stage called G1, cells "monitor" their environment, and when the requisite signals are received, the cells synthesize RNA and proteins to induce growth. When conditions are right, cells enter the S stage of the cell cycle and "commit" to DNA synthesis and replicate their chromosomal DNA. Finally, in the G2 phase, cells continue to grow and prepare for mitosis.
Analysis of Cellular DNA Content
BD Biosciences offers a wide variety of reagents to study the cell cycle. Reagents include DNA dyes such as propidium iodide (PI) and 7-aminoactinomycin D (7-AAD). In addition, the BD Cycletest Plus reagent kit includes PI and other reagents to degrade proteins and RNA to allow more precise DNA measurement. The samples are subsequently analyzed using flow cytometry to assess ploidy, identify abnormal DNA stemlines, and estimate the DNA index (DI) and cell cycle phase distributions of stemlines. During the cell cycle phases, DNA levels change, facilitating the use of DNA dyes such as 7-AAD to generate characteristic cellular DNA content profiles (see the figure below). As cells go through the phases of the cell cycle, proteins such as histone H3 Ser28 become modified or change in expression. To facilitate DNA replication the histone is modified, opening the chromatin to allow entry of replication machinery. To further support the study of cell cycle, BD Biosciences carries antibodies to these proteins to use for imaging or flow cytometry applications.
|DNA||Propidium Iodide (PI), 7-aminoactinomycin D (7-AAD)||Interaction into DNA double strands||Flow cytometry||Fixed, permeabilized, and for live/dead discrimination in intact cells|
|Cell Dyes||BD Horizon™ violet proliferation dye 450 (VPD450)||Diffuses into live cells and is hydrolyzed by intracellular non-specific esterases to become fluorescent products.||Flow cytometry||Live proliferating cells|
|Newly Synthesized DNA||BrdU and antibodies to BrdU||Bromodeoxyuridine replaces thymidine (T) in dividing DNA. It is then detected by antibodies to BrdU.||Flow cytometry, cell imaging, immunohistochemistry||Fixed and permeabilized cells, treated tissues (cell imaging, immunohistochemistry only)|
|Protein Level||Antibodies to Ki67, PCNA||Levels increase as a result of proliferation.||Flow cytometry, bioimaging, immunohistochemistry, Western blot||Fixed cells, tissues, and extracts|
|Protein Level||Antibodies to cyclins, retinoblastoma (Rb), other cell cycle markers||Levels go up and down at different stages of the cell cycle.||Flow cytometry, bioimaging, immunohistochemistry, Western blot||Fixed cells, tissues, and extracts|
|Protein Modification||Antibodies to phosphorylated histone H3, cyclin dependent kinases (cdk)||Proteins become phosphorylated as a result of proliferation or changes to the cell cycle.||Flow cytometry, bioimaging, immunohistochemistry, Western blot|
|BD™ CBA (for quantitative detection)||Fixed cells, tissues, and extracts|
New tool to determine cell divisions
Cell proliferation can occur in response to many stimuli such as cytokine exposure or a variety of other processes. BD has a new product to help researchers study cell proliferation.
BD Biosciences offers BD Horizon™ Violet Proliferation Dye 450 for the detection of cell proliferation with the violet laser, which facilitates the use of larger panels. This allows the determination of more data from limited samples using multicolor flow cytometry.
VPD450 is a nonfluorescent esterified dye. The ester group allows the dye to enter the cell. Once the dye is inside the cell, esterases cleave off the ester group to convert the dye into a fluorescent product and trap it inside the cell. With each replication event the amount of dye in the cell is decreased, leading to a characteristic pattern.
Measurement of Cell Proliferation with BrdU
BD Biosciences carries a series of antibodies and kits designed for the detection of proliferating cells by measurement of bromodeoxyuridine (BrdU), an analog of the DNA precursor thymidine used to measure de novo DNA synthesis.
During the S phase of the cell cycle (DNA synthesis) BrdU is incorporated into the newly synthesized DNA and can be readily detected by anti-BrdU specific antibodies. BD antibodies and kits designed for the detection of BrdU are available for both intracellular flow cytometry and immunohistochemistry and include BD Horizon™ V450 and PerCP-Cy™5.5 formats.
In addition to DNA increases, levels of certain proteins also rise as a result of cell proliferation. For example, Ki67 is an antigen that is expressed in the nucleus of dividing cells. However, during the G0 phase of the cell cycle it is not detected. Ki67 can be combined with other proliferation markers such as BrdU and VPD450 for added confidence. These markers can also be combined with cell surface and other types of markers to gain additional information about cell subsets and their signaling pathways.
The importance of tissue 天津体育彩票网ostasis
As cells become damaged or are no longer needed, they undergo apoptosis or programmed cell death, a normal physiological process that occurs during embryonic development and tissue 天津体育彩票网ostasis maintenance.
Apoptosis is an organized process that signals cells to self destruct for cell renewal or to control aberrant cell growth. Apoptosis controls the orderly death of damaged cells, whereas necrosis occurs as a result of tissue damage, causing the loss of both damaged and surrounding cells.
The apoptotic process is characterized by certain morphological features. These include changes in the plasma membrane (such as loss of membrane symmetry and loss of membrane attachment), a condensation of the cytoplasm and nucleus, protein cleavage, and internucleosomal cleavage of DNA. In the final stages of the process, dying cells become fragmented into "apoptotic bodies" and consequently are eliminated by phagocytic cells without significant inflammatory damage to surrounding cells.
However, some cell types do not display characteristic features of apoptosis. In those cases multiple aspects of apoptosis might need to be analyzed to confirm the mechanism of cell death.
To support this spectrum of requirements, BD Biosciences offers a full range of apoptosis detection tools and technologies for measuring indicators at different stages across the apoptotic process. BD Biosciences tools use multiple methodologies including flow cytometry, bioimaging, and microscopy (for live and fixed cell analysis) as well as ELISA, IHC, Western blot, and spectrofluorometry.
Annexin V–A Key Protein in Apoptosis Signaling
Changes in the plasma membrane are one of the first characteristics of the apoptotic process detected in living cells. Apoptosis can be detected by the presence of phosphatidylserine (PS), which is normally located on the cytoplasmic face of the plasma membrane. During apoptosis PS translocates to the outer leaflet of the plasma membrane and can be detected by flow cytometry and cell imaging through binding to fluorochrome-labeled Annexin V when calcium is present.
BD Biosciences offers Annexin V in several common formats such as FITC, PE, and BD Horizon™ V450 for the violet laser. With the addition of these new formats, more complex assays can be developed to look at apoptosis within heterogeneous cell subsets.
Since intracellular Annexin V is also exposed if the plasma membrane is compromised, a membrane-impermeant dye such as 7-AAD is commonly used to distinguish between apoptotic and dead cells to exclude the dead cells. The populations of cells that are stained with Annexin V only represent the apoptotic cell populations.
Tools to streamline apoptosis research
There are many apoptosis triggers including certain cytokines, protein-protein interactions, and chemicals. Once apoptosis starts, changes in the mitochondria membrane potential can be measured by flow cytometry using the BD™ MitoScreen (JC-1) flow cytometry kit.
Increases in mitochondrial membrane potential lead to increased mitochondrial membrane permeability and the release of soluble proteins such as cytochrome c and pro-caspases.
Caspases are a series of proteases activated upon cleavage at aspartate residues during earliest stages of apoptosis. Active caspases can then cleave many proteins including Poly-ADP ribose polymerase (PARP) and other caspases.
DNA fragmentation is one of the last phases in apoptosis resulting from the activation of endonucleases during the apoptotic process. There are several established methods for the study of DNA fragmentation including isolation and separation of DNA fragments by agarose gel electrophoresis and end labeling.
The BD™ APO-BrdU kit uses end labeling or the terminal deoxynucleotidyl transferase (TdT) nick end labeling (TUNEL method) to support the study of DNA fragmentation. In this assay, TdT catalyzes a template-independent addition of bromolated deoxyuridine triphosphates (Br-dUTP) to the 3'-hydroxyl (OH) termini of double- and single-stranded DNA. After the Br-dUTP is incorporated, these terminal sites of double- and single-stranded DNA are identified using flow cytometry by staining cells with labeled anti-BrdU. In contrast, the BrdU proliferation assay incorporates BrdU into newly synthesized DNA, into sites of DNA strand breaks.
|Feature Measured||Assays||Key Features|
|Plasma Membrane Alterations
|Annexin binding assay
|Mitochondrial Changes||BD MitoScreen Kit||Fast, easy, single cell resolution by flow cytometry or fluorescent microscopy|
|Caspase Activation||Caspase Activity Assay Kits and Reagents||Quick and easy, uses spectrofluorometry|
|Active Caspase-3 immunoassays||ELISA, flow cytometry, or Western blot|
||Works with adherent cells, single cell resolution in conjunction with cell cycle analysis by flow cytometry|
With an overwhelming number of available techniques and products, selecting the most appropriate method is often difficult. To help make this choice easier, the overview above summarizes commercially available assays from a biological perspective.
Measurement of Cleaved Caspases and PARP
Caspases are important initiators of apoptosis. One of the earliest and most consistently observed characteristics of apoptosis is the activation of a series of cytosolic proteases, called caspases. When apoptosis is activated, caspases cleave multiple protein substrates en masse, which leads to the loss of cellular structure and function, and ultimately results in cell death. In particular, caspases -8, -9, and -3 have been implicated in apoptosis: caspase-9 in the mitochondrial pathway, caspase-8 in the Fas/CD95 pathway, and caspase-3 more downstream, activated by multiple pathways.
BD Biosciences carries a variety of reagents to measure caspases, particularly caspase-3. They include antibodies directed exclusively against the active form of the caspase. These antibodies are available in a variety of formats and can be used for flow cytometry, imaging, ELISA, and Western blot.
BD Biosciences offers a range of tools for caspase activity assays from individual fluorogenic peptide substrates and inhibitors, to kits, to ready-to-use assay plates. All are based on the use of synthetic tetrapeptide substrates that are designed such that proteolytic cleavage by active human or mouse caspases results in release of a fluorophore or chromophore. The individual synthetic tetrapeptide substrates, together with the caspase inhibitors and active caspase enzymes, offer flexibility in the experimental design of a caspase activity assay.
Obtain the complete picture
In addition to caspases and Annexin V, there are several other proteins important for the study of apoptosis, including the Bcl-2 family, tumor necrosis factor receptor (TNFR) family, PARP, and other signaling molecules.
Bcl-2 family members, identified by the presence of conserved BCL2 homology (BH3) domains, are versatile key regulators of apoptosis. Bcl-2, for example, protects cells from apoptosis by associating with the mitochondrial membrane and preventing the release of cytochrome c from the mitochondria. In contrast other Bcl-2 family members such as Bax promote apoptosis. Increased levels of Bcl-2 have been reported in cancer.
The TNFR family contains many members, including CD95, that can be divided into three major groups based on structure. Signaling through the TNFR pathway leads to apoptosis.
PARPs are DNA repair enzymes that are activated by DNA strand breaks. Cleavage of PARP by caspase-3 into 24- and 89-kDa fragments inactivates the PARP enzyme.
BD Biosciences carries antibodies specific for cleavage products of PARP that are useful markers of apoptosis. These antibodies are available in a variety of formats and can be combined with other markers to gain additional information about the cell.
Simultaneous Studies of Apoptosis, Cell Cycle, and DNA Damage
Apoptosis and cell proliferation assays are particularly useful for basic cancer research and drug discovery. Comparing data across different experiments can be challenging due to variability introduced by sample handling, timing, and variability within the sample.
Multicolor flow cytometry addresses these challenges and is an excellent tool to study apoptosis and cell proliferation. Relevant markers can be combined with cell phenotyping markers to look at events within subpopulations of cells. Antibodies to phosphoproteins can be used to examine phosphorylation events.
CD4-enriched mouse splenocytes were cultured with anti-CD3/CD28, IL-2, and IL-4
Cell cycle analysis of a population stained for incorporated BrdU and total DNA levels (7-AAD).
The use of VPD450 to correlate cell proliferation with IL-2 production.
Cell cycle analysis of HeLa cells treated with aphidicolin.
Radio frequency dose-dependent apoptosis, necrosis, and cell death monitored by Annexin V - BD Horizon V450.
Flow cytometric analysis of apoptotic and non-apoptotic populations using anti-active caspase-3 antibodies.
In this experiment, Jurkat cells were treated with camptothecin, a potent inhibitor of topoisomerase I and apoptosis inducer.
- Cell Cycle Analysis Using the BD BrdU FITC Assay on the BD FACSVerse™ System
- Detection of Apoptosis Using the BD Annexin V FITC Assay on the BD FACSVerse™ System
- Multiple Methods for Detecting Apoptosis on the BD Accuri™ C6 Flow Cytometer
- Technique for Loading Cells with BD Horizon™ Violet Proliferation Dye 450 (VPD450)
Product Information Sheets
- Annexin V Reagents
- Apoptosis, DNA Damage, and Cell Proliferation Kit
- BD Biosciences Nucleic Acid Dyes
- BD FACS™ Pre-Sort Buffer
- BD Horizon™ Cell Proliferation Dyes
- BD Horizon™ Fixable Viability Stain 450
- BD Horizon™ Fixable Viability Stain (FVS) Reagents
- BD Pharmingen™ Proliferation Kits for cell cycle analysis
- Annexin V with Cell Surface Antibody Staining for Suspension Cells
- Annexin V Staining of Adherent Cells for Flow Cytometry
- Annexin V Staining Protocol
- APO-BRDU™ Procedure (TUNEL Assay for Flow)
- APO-DIRECT™ Procedure (TUNEL Assay for Flow)
- Cell Death Induced by Camptothecin
- Cell Death Induced by Staurosporine
- Induction of Apoptosis Using Anti-Human Fas (CD95), Clone DX2
- Induction of Apoptosis Using Anti-Mouse Fas (CD95), Clone Jo2
For Research Use Only. Not for use in diagnostic or therapeutic procedures.