Cytokines: Intracellular Staining Reagents
For a protocol on Intracellular Immunofluorescent Staining for Flow Cytometry, please visit www.ebioscience.com/ebioscience/appls/FCI.htm.
IL-21, which is structurally-related to IL-2, IL-4, and IL-15, has a variety of roles within lymphoid lineages, including promotion of CD8 T cell function and inhibition of IgE production in B cells. IL-21 is produced by activated T cells, and it influences proliferation of T and B cells and cytolytic activity of NK cells. Within the NK cell lineage, IL-21 enhances NK cell maturation from human bone marrow progenitors and activates peripheral NK cells. In the mouse, IL-21 has the contrasting effect of inhibiting the IL-15-promoted expansion of resting NK cells, while also inducing a dramatic increase in NK cell size, granularity, loss of NK1.1, and upregulation of NKG2/CD94 complex – correlating with increased cytotoxicity. Activated mouse NK cells treated with IL-21 have been found to have heightened NK cell-mediated cytotoxicity/antitumor immunity and IFN-γ production (over IL-15 treatment alone). IL-21 – treated mice display enhanced capacity to inhibit liver and lung tumor metastases, in an NK- and perforin- dependent manner. Moreover, IL-21 has been found to enhance tumor rejection in vivo through an NKG2D-dependent mechanism.
References: Parrish-Novak, J., et al. 2000. Nature. 408(6808):57-63. Parrish-Novak, J., et al. 2002. J Leukoc Biol. 72(5):856-63.
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Human IL-21: Intracellular staining of unstimulated (left) or PMA/ionomycin/ monensin stimulated (right) human PBMCs with FITC anti-human CD4 (RPA-T4) (cat. 11-0049) and Alexa Fluor® 647 anti-human IL-21 (eBio3A3-N2) (cat. 51-7219). Cells in the lymphocyte gate were used for analysis.
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Granulysin, a protein involved in the cytolytic pathway, is a member of the saposin-like protein (SAPLIP) family. Two proteins 9 and 15kDa are thought to be post translation modified versions of the 15kDa protein with a change in confirmation. Granulysin is found in NK and activated T cells and the expression is confined to the cytolytic granules which also contains perforin and granzyme. Granulysin with perforin are suspected to function synergistically to kill bacteria and induce apoptosis in nucleated cells.
References: Gansert, J.L., et al. 2003. J Immunol. 170(6):3154-61. Hanson, D.A., et al. 1999. Mol Immunol. 36(7):413-22. Ochoa, M.T., et al. 2001. Nat Med. 7(2):174-9. Stegelmann, F., et al. 2005. J Immunol. 175(11):7474-83. Stenger, S., et al. 1998. Science. 282(5386):121-5. Stenger, S., et al. 1998. Science. 282(5386):121-5.
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Human Granulysin: Staining of normal human peripheral blood mononuclear cells with FITC anti-human CD56 (MEM188) (cat. 11-0569) then fixed and permeabilized and stained with PE Mouse IgG1, K Iso Cntrl (cat. 12-4714) (left) or PE anti-human Granulysin (eBioDH2) (cat. 12-8828) (right). Cells in the lymphocyte gate were used for analysis.
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IL-13 is a cytokine produced mainly by Th2 cells, but also by antigen-primed CD8 T cells. IL-13 has a strong involvement in allergic inflammation and parasitic clearing and in cancer models has been shown to have either inhibitory or stimulatory activity depending on the tumor. In humans, IL-13 is found to play a role in isotype switching in B cells. IL-13 is implicating in down modulating macrophage activity, through the reduction of pro-inflammatory cytokines (IL-1, IL-6, IL-8, IL-10, IL-12).
References: Miyahara, N., et al. 2004. J. Immunol. 172: 2539-2558. McKenzie, A.N., et al. 1993. J Immunol. 150(12):5436-44. Doherty, T.M., et al. 1993. J Immunol. 151(12):7151-60. Terabe, M., et al. 2000. Nat Immunol. 1(6):515-20. Grunig, G., et al. 1998. Science. 282(5397):2261-3.
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Mouse IL-13: Mouse splenocytes were activated, rested, and reactivated with PMA/Ionomycin in the presence of monensin for 5 hours. Cells were stained with FITC
anti-mouse CD4 (RM4-5) (cat. 11-0042), fixed and permeabilized, then stained with PE anti-mouse IL-13 (eBio13A) (cat. 12-7133) (left plot);
IL-13 staining was abrogated by pre-blocking with ligand (right plot).
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Table 1: Mouse Intracellular Staining Reagents
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Mouse Intracellular Staining Reagents
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| Antigen |
Clone |
SKU |
Formats |
| CD152/CTLA4
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UC10-4B9
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1522
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Purified, FG Purified, Biotin, PE
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| GM-CSF
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MP1-22E9
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7331
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Purified, FG Purified, FITC, PE
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| Granzyme B
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16G6
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8822
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Purified, Biotin, FITC, PE, Alexa Fluor® 647
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| IFN-γ
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XMG1.2
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7311
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Purified, FG Purified, Biotin, FITC, PE, APC, PE-Cy7, Cy5, Alexa Fluor® 488,
Alexa Fluor® 647, Pacific Blue®
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| IL-1α
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ALF-161
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7011
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Purified, FG Purified, FITC, PE
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| IL-2
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JES6-5H4
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7021
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Purified, FG Purified, Biotin, FITC, PE, APC, Alexa Fluor® 488, Alexa Fluor® 647, Pacific Blue®
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| IL-4
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11B11
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7041
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Purified, FG Purified, PE, APC, Alexa Fluor® 488, Alexa Fluor® 647
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BVD6-24G2
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7042
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Purified, Biotin, FITC, PE-Cy7
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| IL-5
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TRFK5
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7052
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Purified, FG Purified, PE
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| IL-6
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MP5-20F3
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7061
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Purified, FG Purified, FITC, PE
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| IL-10
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JES5-16E3
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7101
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Purified, FG Purified, FITC, PE, APC, Alexa Fluor® 488, Alexa Fluor® 647
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| IL-12/IL-23 p40
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C17.8
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7123
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FG Purified, Biotin, PE, Alexa Fluor® 647
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| IL-13
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eBio13A
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7133
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Purified, FG Purified, PE, Alexa Fluor® 647
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| IL-17A
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eBio17B7
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7177
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FG Purified, Biotin, FITC, PE, APC, Alexa Fluor® 488, Alexa Fluor® 647
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| IL-17F
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eBio18F10
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7471
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PE, Alexa Fluor® 488, Alexa Fluor® 647
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| Perforin
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eBioOMAK-D
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9392
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Purified, FITC, PE, APC
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| TNF-α
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MP6-XT22
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7321
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Purified, FG Purified, FITC, PE, APC, Cy5, Alexa Fluor® 488, Alexa Fluor® 647,
Pacific Blue®
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TN3-19
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7423
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Purified, FG Purified, FITC, PE, PE-Cy7
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| Alexa Fluor® and Pacific Blue® are registered trademarks of and licensed under patents assigned to Molecular Probes, Inc. for research use only. |
Table 2: Human Intracellular Staining Reagents
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Human Intracellular Staining Reagents
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| Antigen |
Clone |
SKU |
Formats |
| CD152/CTLA4
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14D3
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1529
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Purified, FG Purified, Biotin, PE
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eBio20A
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1528
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Purified, FG Purified, Biotin, PE
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| Granzyme B
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eBioGrB
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8899
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PE
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| Granulysin
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eBioDH2
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8828
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PE
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| IFN-α2
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eBio225.c
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8317
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FITC, PE, APC
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| IFN-γ
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4S.B3
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7319
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Purified, Biotin, FITC, PE, APC, PE-Cy7, Alexa Fluor® 488, Alexa Fluor® 647, Pacific Blue®
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| IL-1α
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CRM8
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7019
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Purified, FG Purified, FITC
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364/3B3-14
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7118
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Purified, FITC, PE
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| IL-1β
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CRM56
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7018
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Purified, FG Purified, FITC, PE
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| IL-2
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MQ1-17H12
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7029
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Purified, FITC, PE, APC
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| IL-4
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8D4-8
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7049
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Purified, PE, APC, PE-Cy7, Alexa Fluor® 488, Alexa Fluor® 647
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MP4-25D2
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7048
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Purified, FG Purified, Biotin, FITC, PE, APC
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| IL-5
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JES1-5A10
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7059
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FG Purified, Biotin, PE
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TRFK5
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7052
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Purified, FG Purified, PE
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| IL-6
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MQ2-13A5
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7069
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Purified, FG Purified, FITC, PE
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| IL-10
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JES3-9D7
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7108
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Purified, FG Purified, PE, Alexa Fluor® 488, Alexa Fluor® 647, Pacific Blue®
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| IL-12/IL-23 p40
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C8.6
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7129
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Purified, FG Purified, Biotin, PE, Alexa Fluor® 488, Alexa Fluor® 647, Pacific Blue®
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eBioHP40
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7235
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Alexa Fluor® 647
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| IL-13
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PVM13-1
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7139
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Purified, FG Purified, FITC
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| IL-17A
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eBio64CAP17
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7178
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Purified, FG Purified, PE, Alexa Fluor® 647
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eBio64DEC17
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7179
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Biotin, FITC, PE, Alexa Fluor® 488, Alexa Fluor® 647
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| Perforin
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dG9
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9994
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Purified, FITC, PE
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| TNF-α
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MAb11
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7349
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Purified, Biotin, FITC, PE, APC, PE-Cy7, Alexa Fluor® 488, Alexa Fluor® 647, Pacific Blue®
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| TNF-β
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359-81-11
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7327
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Biotin, PE, APC
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| Alexa Fluor® and Pacific Blue® are registered trademarks of and licensed under patents assigned to Molecular Probes, Inc. for research use only. |
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