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Cell & Tissue Analysis

Apoptosis

Since there is no single parameter that defines programmed cell death, a combination of techniques is recommended for reliable detection of apoptosis. Using timed inductions and comparing relationships between cell populations expressing multiple markers, it is possible to estimate within a given model the relative orders in which different components of an apoptotic process become evident. We have looked at a number of reagents using Jurkat cells induced with 10 M camptothecin for time periods of 0 to 4 hours. The chart below refers to when changes were observed, based on expression patterns relative to the appearance of annexin V staining and to the loss of membrane integrity (PI, SYTOX® Green dye).

Apoptosis Timeline

  • Time after induction  Cell function changes Reagents used
    start













    end
    		Mitochondrial
    membrane potential    		MitoTracker® Red
    MitoProbe™ DiOC2(3)
    Assay Kit
    MitoProbe™ JC-1 Assay Kit
     Mitochondrial
    transition pore opening    		Image-iT™ LIVE Mitochondrial Transition Pore Assay Kit
    MitoProbe™ Transition Pore Assay Kit
     Phosphatidylserine translocation to outer membraneAnnexin
    Caspase activityCaspase Kits
    Metabolic activityVybrant® Cell Metabolic Assay Kit
    DNA condensationHoechst nucleic acid stain
    Plasma membrane integrityPropidium iodide
    SYTOX® Green dye
    DNA fragmentationAPO-BrdU TUNEL Assay Kit
    AntiBrdU Antibodies

Table 1

Target Antibody type Clone/PAD Western blotting ELISA Immuno- precipitation Immuno- histo- chemistry Immuno- fluorescence Flow cytometry Cat. no.
Annexin II Mouse IgG1 Z014       03-4400
ASPP1 Mouse IgG1 kappa 54.2       39-7100
Specific for apoptosis-stimulating protein of p53 protein 1, p53 binding protein 1, PPP1R13B.
ASPP2 (C-term) Mouse IgG1 kappa DX54.10     39-7000
Specific for apoptosis-stimulating protein of p53 protein 2, p53 binding protein 2, 53BP2, Bcl2-binding protein (Bbp), PPP1R13A.
ASPP (C-term) Rabbit polyclonal             40-9600
Specific for inhibitor of apoptosis-stimulating protein of p53, RelA-associated inhibitor, NFkB-interacting protein 1, protein phosphatase 1 regulatory subunit 13-like, PPP1R13L protein.
BAG1 Mouse IgG1 kappa 10B6H7   37-4000
Specific for human BAG-1, BAG-1M, and BAG-1L proteins.
Li, J. et al. Oncogene 24, 5510[endash]5520 (2005)(WB)
BAK Mouse IgG2a kappa ZB001         39-4100Z
Specific for Bcl-2 homologous antagonist/killer, Bcl-2-like 7 protein, cell death inhibitor 1 protein.
Bax Mouse IgG1 2D2      33-6400
18-0218
Specific for the 21 kDa Bax protein.
Bax Mouse IgG1 6A7     33-6600
Specific for the 21 kDa Bax protein.
Bcl-2 Mouse IgG1 kappa Bcl-2-100   13-8800
18-0193Z
Specific for the human Bcl-2 protein.
Bcl-2 Mouse IgG1 10C4         33-6100
Specific for mouse and rat Bcl-2 proteins.
Bcl-3 (mid) Rabbit polyclonal ZMD.364         38-3100
Reacts with the ~60 kDa human Bcl-3 protein.
Bcl-3 (C-term) Rabbit polyclonal ZMD.365             38-3200
Reacts with the ~60 kDa human Bcl-3 protein.
Bcl-XL Mouse IgG2a 2H12                 33-6300
18-0217
Bim Rabbit polyclonal ZMD.397           38-6500
Specific for the ~23 kDa human and mouse Bim proteins.
BrdU Mouse IgG1 kappa PRB-1       A21300
A21303
A21304
A21305
Caspase-3 Mouse IgG2a 4-1-18         35-1600Z
Specific for human caspase 3.
Cytochrome c (native) Mouse IgG1b kappa 6H2.B4         33-8200
Specific for native cytochrome c, and does not cross-react with denatured cytochrome c.
Cytochrome c (denatured) Mouse IgG2b kappa 7H8.2C12               33-8500
Cytochrome c (Alexa Fluor 488 conjugate) Mouse IgG               S35115
FADD Mouse IgG1 3A4D4            35-8000
Specific for the ~24 kDa FADD protein.
iASPP (C-term) Rabbit polyclonal ZMD.517              40-9600
Specific for the iASPP (inhibitor of apoptosis stimulating protein of p53, RelA-associated inhibitor, NFkB-interacting protein 1, protein phosphatase 1 regulatory subunit 13-like, PPP1R13L) protein.
Lamin B1 Mouse IgG1 kappa L-5                  33-2000
Specific for the 68 kDa lamin B1 isoform; reactivity with other lamin isoforms has not been detected.
Lamin B2 Mouse IgG1 kappa E-3                  33-2100
Specific for the 66 kDa lamin B2 isoform; reactivity with other lamin isoforms has not been detected.
Nix Mouse IgG1 kappa ZN002                39-3300
Specific for the Nix (Nip3-like protein X, BNIP3a, BNIP3L) protein
p53 (AIP1) Rabbit polyclonal Z15.MP               52-3587
PARP Mouse IgG1 kappa C-2-10                33-3100
Specific for the 116 kDa full-length PARP protein, as well as its 85 kDa cleavage fragment. The immunoreactive epitope is contained within the C-terminal region of the 46 kDa DNA-binding domain of the PARP protein.
RCAS1 Mouse IgG1 kappa ZR001                 39-2300
Specific for the RCAS1 protein.
Smac/DIABLO Mouse IgG1 17-1-87                  35-6600
Specific for Smac/DIABLO, a 29 kDa mammalian mitochondrial protein that regulates apoptosis by binding apoptosis inhibitor proteins (IAPs).
Survivin Mouse IgG1 3F5H5             37-2000
Specific for the ~16 kDa survivin protein.
Tid1 Rabbit polyclonal ZMD.513               40-9200
Specific for human Tid1 (tumorous imaginal discs protein, Tid56 and DnaJ (Hsp40) homolog, subfamily A member 3, DNAJA3) protein.