Description
The DX2 monoclonal antibody specifically binds to the human Fas antigen (also called APO-1). This 45 kDa type I transmembrane glycoprotein was designated as CD95 at the Fifth HLDA Workshop. Fas is a member of the TNF-receptor superfamily and is also known as Tumor necrosis factor receptor superfamily member 6 (TNFRSF6). It is differentially expressed on a variety of normal and neoplastic cells. These include some undifferentiated thymocytes, and activated T and B lymphocytes, natural killer (NK) cells, monocytes, neutrophils, fibroblasts, and cell lines. CD95 is preferentially expressed on CD45RO-positive memory T lymphocytes and γ/δ T lymphocytes. The Fas/CD95 antigen is a polypeptide that plays a role in the programmed sequence of events leading to cell death, termed apoptosis. Crosslinking CD95 with DX2 antibody delivers an apoptotic signal indicating that DX2 recognizes a functional epitope of the CD95 antigen.
Format
- FormatNA/LE
Other Formats→
- APC
- BB515
- BB700
- BUV395
- BUV615
- BUV737
- BV421
- BV480
- BV510
- BV650
- BV711
- BV786
- Biotin
- FITC
- PE
- PE-CF594
- PE-Cy™5
- PE-Cy™7
- PerCP-Cy™5.5
- Purified
- V450
Suggested Companion Products
FITC Goat Anti-Mouse IgG/IgM PolyclonalRUO 0.5mgCat No: 555988
Purified NA/LE Mouse IgG1 κ Isotype Control RUO 0.5mgCat No: 554721
APO-DIRECT™ Kit RUO 50TestsCat No: 556381
APO-BRDU™ Kit RUO 60TestsCat No: 556405
FITC Annexin V RUO 100TestsCat No: 556420
Stain Buffer (FBS) RUO 500mLCat No: 554656
Stain Buffer (BSA) RUO 500mLCat No: 554657
Lysing Buffer RUO 100mLCat No: 555899
Lysing Solution 10X Concentrate IVD 100Cat No: 349202
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Preparation and Storage
Store undiluted at 4°C.The monoclonal antibody was purified from tissue culture supernatant or ascites by affinity chromatography.This preparation contains no preservatives, thus it should be handled under aseptic conditions.
Product Notices
- Since applications vary, each investigator should titrate the reagent to obtain optimal results.
- An isotype control should be used at the same concentration as the antibody of interest.
- Species testing during development may have been performed with a different format of the same clone.Selected applications have been tested for cross-reactivity.
- Please refer to www.bdbiosciences.com/pharmingen/protocols for technical protocols.
Investigators are advised that the following procedure is not routinely tested for this material.
Induction of Apoptosis Using Purified Mouse Anti-Human CD95 (Clone DX2) [MN 555670]
Additional Materials Needed:
Positive control cell line (e.g., Daudi, HPB-ALL, Jurkat)
Recombinant Protein G (rProt G) (SIGMA, Cat. No. P4689)
96-well microtiter plate
IMDM or RPMI 1640 medium with 10% heat-inactivated fetal bovine serum (FBS), 1% L-glutamine, 1% antibiotics (FBS medium)
Procedure:
1. Maintain cells in culture with 10% FBS medium; change the medium one day before starting the induction of apoptosis. Harvest and pellet the cells, resuspend in 10% FBS medium at a density of 0.5-1.0 x 10^6 cells/ml.
2. In a 96-well microtiter plate, add 2.5 µg/50 µl CD95 NA/LE™, 0.5 µg/50 µl rProt G, 0.5-1.0 x 10^5 cells and 10% FBS medium to a total volume of 200 µl. Negative controls should consist of: 1) 0.5-1.0 x 10^5 cells with 10% FBS medium alone (200 µl total volume), and 2) 0.5-1.0 x 10^5 cells with 0.5 µg/50 µl rProtG and 10% FBS medium.
3. Incubate the 96-well plate at 37°C, 5% CO2, for 12-24 hours. Traditionally, apoptosis has been observed by light microscopy, MTT, or gel electrophoresis (DNA fragmentation). Investigators may be interested in several products offered by BD Biosciences that may be used for the detection of apoptotic cells by flow cytometry: ApoDirect™ Kit (Cat. No. 556381), ApoBRDU™ Kit (Cat. No. 556405) and Annexin V-FITC (Cat. No. 556420 or 556419). Because these methods vary in sensitivity, it may be necessary to titer the rProtG and/or CD95 NA/LE™ to obtain optimal results.
Notes:
The suggestions given in this procedure are based on conditions that were found to be optimal for the induction of apoptosis by anti-human CD95 (Fas). Studies have shown that the addition of protein G can significantly enhance the efficiency of DX2 in this type of functional assay.It is important to note that there is a great deal of variation between cell lines in the level of apoptosis that can be induced through the Fas receptor. It has been reported that, although some cells express the Fas antigen, they do not necessarily undergo Fas-mediated apoptosis (i.e., the Fas antigen expressed may be anti-Fas sensitive or insensitive). Daudi, Jurkat, and HPB-ALL cells are good positive controls as they are strongly induced by anti-Fas to undergo apoptosis.
