PE-Cy™7 Mouse Anti-Human TNF
Clone MAb11 (RUO)
- Brand BD Pharmingen™
- Alternative Name Tumor necrosis factor alpha; TNF-a; TNF-α; TNFSF2; Cachectin
- Vol. Per Test 5 µl
- Isotype Mouse IgG1, κ
- Reactivity Human (QC Testing) Rhesus, Cynomolgus, Baboon (Tested in Development)
Intracellular staining (flow cytometry) (Routinely Tested)
ELISA (Tested During Development)
- Immunogen Recombinant Human TNF
- Storage Buffer Aqueous buffered solution containing BSA and ≤0.09% sodium azide.
- Regulatory Status RUO
Regulatory Status Legend
The MAb11 monoclonal antibody specifically binds to human tumor necrosis factor (TNF, also known as TNF-α) protein. TNF is an efficient juxtacrine, paracrine and endocrine mediator of inflammatory and immune functions. It regulates the growth and differentiation of a variety of cell types. TNF is cytotoxic for transformed cells when in conjunction with IFN-γ. It is secreted by activated monocytes/macrophages and other cells such as B cells, T cells and fibroblasts. The immunogen used to generate the MAb11 hybridoma was recombinant human TNF. The MAb11 antibody has been reported to crossreact with Rhesus Macaque TNF.
- Format PE-Cy™7
- Excitation Source Blue 488 nm,Green 532 nm,Yellow/Green 561 nm
- Excitation Max 496 nm
- Emission Max 785 nm
PE-Cy™7 is a tandem fluorochrome that combines PE and a cyanine dye. PE-Cy7 conjugated reagents are as bright as PE conjugates. PE-Cy7 is particularly sensitive to photo-induced degradation, resulting in loss of fluorescence and changes in fluorescence spillover. Extreme caution must be taken to avoid light exposure and prolonged exposure to paraformaldehyde fixative. Fixed cells should be analyzed within 4 hours of fixation in paraformaldehyde or transferred to a paraformaldehyde-free buffer for overnight storage.
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Preparation and Storage
Store undiluted at 4°C and protected from prolonged exposure to light. Do not freeze.
The monoclonal antibody was purified from tissue culture supernatant or ascites by affinity chromatography.
The antibody was conjugated with PE-Cy7 under optimum conditions, and unconjugated antibody and free PE-Cy7 were removed.
- 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).
- An isotype control should be used at the same concentration as the antibody of interest.
- Source of all serum proteins is from USDA inspected abattoirs located in the United States.
- 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.
- PE-Cy7 is a tandem fluorochrome composed of R-phycoerythrin (PE), which is excited by 488-nm light and serves as an energy donor, coupled to the cyanine dye Cy7, which acts as an energy acceptor and fluoresces maximally at 780 nm. PE-Cy7 tandem fluorochrome emission is collected in a detector for fluorescence wavelengths of 750 nm and higher. Although every effort is made to minimize the lot-to-lot variation in the efficiency of the fluorochrome energy transfer, differences in the residual emission from PE may be observed. Therefore, we recommend that individual compensation controls be performed for every PE-Cy7 conjugate. PE-Cy7 is optimized for use with a single argon ion laser emitting 488-nm light, and there is no significant overlap between PE-Cy7 and FITC emission spectra. When using dual-laser cytometers, which may directly excite both PE and Cy7, we recommend the use of cross-beam compensation during data acquisition or software compensation during data analysis.
- 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.
- Warning: Some APC-Cy7 and PE-Cy7 conjugates show changes in their emission spectrum with prolonged exposure to formaldehyde. If you are unable to analyze fixed samples within four hours, we recommend that you use BD™ Stabilizing Fixative (Cat. No. 338036).
- For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
- Cy is a trademark of GE Healthcare.
- 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/us/s/resources for technical protocols.
Immunofluorescent Staining and Flow Cytometric Analysis: The MAb11 antibody is useful for intracellular immunofluorescent staining and flow cytometric analysis to identify and enumerate TNF producing cells within mixed cell populations. The FITC-, PE-, APC-, and PE-Cy7-conjugated MAb11 antibodies (Cat. No. 554512, 554513 , 554514, and557647, respectively) are especially suitable for these studies (see image). For optimal immunofluorescent staining with flow cytometric analysis, this anti-cytokine antibody should be used at 5 µl/test The staining technique and use of blocking controls are described in detail by C. Prussin and D. Metcalfe.3 For specific methodology, please visit our website, http://www.bdbiosciences.com/us/s/resources, and go to the protocols section under "Cytokines (Intracellular Staining)" or "Intracellular Flow".
ELISA Detection: The biotinylated MAb11 antibody (Cat. No. 554511) can be used as the detection antibody in a sandwich ELISA for measuring human TNF protein levels in conjunction with clone MAb1 (Cat. No. 551220; purified mouse anti-human TNF) as the capture antibody and recombinant human TNF (Cat. No. 554618) as the standard. For specific methodology, please visit the protocols section under "ELISA and ELISPOT" at our website, http://www.bdbiosciences.com/us/s/resources.