BD, 555670, BD Pharmingen™ Purified NA/LE Mouse Anti-Human CD95

Alternative Name: APO-1; FAS; TNFRSF6; APT1; ALPS1A; FAS1; FASTM; FASLG receptor
Reactivity: Human (QC Testing), Rhesus, Cynomolgus, Baboon (Tested in Development)
Isotype: Mouse C3H, also known as C3H/He, C3H/Bi IgG1, κ
Immunogen: Human CD95-transfected L Cells
Application: Flow cytometry (Routinely Tested), Functional assay (Tested During Development)
Concentration: 1.0 mg/ml
Workshop Number: VI C-64
RRID: AB_396023
Storage Buffer: No azide/low endotoxin: Aqueous buffered solution containing no preservative, 0.2µm sterile filtered. Endotoxin level is ≤0.01 EU/µg (≤0.001 ng/µg) of protein as determined by the LAL assay.
Regulatory Status: RUO
Preparation And Storage: 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.
Recommended Assay Procedures: Recommended Assay Procedures 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.
Product Notices: 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 cross-reactivity detected in product development may not have been confirmed on every format and/or application. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.