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Analysis of p38 MAPK (pT180/pY182) in monocytes. Human peripheral blood mononuclear cells (PBMC) were either stimulated with 40 μM Anisomycin for 20 minutes (shaded histogram) or unstimulated (open histogram). The cells were fixed (BD Cytofix™ buffer, Cat. No. 554655) for 10 minutes, then permeabilized (BD Phosflow™ Perm Buffer III, Cat. No. 558050) on ice for at least 30 minutes, and then stained with PE-Cy™7 Mouse anti-p38 MAPK (pT180/pY182) (Cat. No. 560241). Monocytes were selected by scatter profile. Flow cytometry was performed on a BD FACSCanto™ II flow cytometer.
BD™ Phosflow PE-Cy™7 Mouse anti-p38 MAPK (pT180/pY182)
BD™ Phosflow PE-Cy™7 Mouse anti-p38 MAPK (pT180/pY182)
Regulatory Status Legend
Any use of products other than the permitted use without the express written authorization of Becton, Dickinson and Company is strictly prohibited.
Preparation And Storage
Recommended Assay Procedures
Either BD Cytofix™ fixation buffer or BD™ Phosflow Fix Buffer I may be used for cell fixation. Any of the three BD™ Phosflow permeabilization buffers may be used.
Product Notices
- 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.
- 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.
- Source of all serum proteins is from USDA inspected abattoirs located in the United States.
- 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.
- 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.
- 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.
- Species cross-reactivity detected in product development may not have been confirmed on every format and/or application.
- Cy is a trademark of GE Healthcare.
- Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
Companion Products
Activation of the immune and inflammatory responses often involves the recognition of bacterial endotoxin (lipopolysaccharide or LPS). Binding of LPS by monocytes results in the production and release of proinflammatory cytokines, such as IL-1 and TNF. LPS-induced signaling cascades involve members of the Ser/Thr protein kinase family known as the Mitogen Activated Protein Kinases (MAPKs). MAPK signal transduction pathways mediate the effects of various extracellular stimuli on biological processes such as proliferation, differentiation, and death. The p38 MAPKs include p38α (MAPK14), β (MAPK11), γ (MAPK12), and δ (MAPK13). These Ser/Thr kinases are activated by dual phosphorylation on threonine (T) and tyrosine (Y) within the motif Thr-Gly-Tyr located in kinase subdomain VIII. Activation of p38 MAPK is mediated specifically by the MAP Kinase Kinases, MKK3, MKK4, and MKK6. This leads to the activation of multiple transcription factors (NF-κB, ATF-2, Elk-1, and CHOP) that induce expression of many different genes, including proinflammatory cytokine genes. Thus, p38 MAPKs are central kinases in multiple signal transduction pathways.
The 36/p38 (pT180/pY182) monoclonal antibody recognizes the conserved dual phosphorylated site pT180/pY182 of p38α, β, γ, and δ.
Development References (3)
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Brunet A, Pouyssegur J. Identification of MAP kinase domains by redirecting stress signals into growth factor responses. Science. 1996; 272(5268):1652-1655. (Biology). View Reference
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Han J, Lee JD, Bibbs L, Ulevitch RJ. A MAP kinase targeted by endotoxin and hyperosmolarity in mammalian cells. Science. 1994; 265(5173):808-811. (Biology). View Reference
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Winston BW, Chan ED, Johnson GL, Riches DW. Activation of p38mapk, MKK3, and MKK4 by TNF-alpha in mouse bone marrow-derived macrophages. J Immunol. 1997; 159(9):4491-4497. (Biology). View Reference
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