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The monoclonal antibody was purified from tissue culture supernatant or ascites by affinity chromatography. The antibody was conjugated with R-PE under optimum conditions, and unconjugated antibody and free PE were removed. Store undiluted at 4°C and protected from prolonged exposure to light. Do not freeze.
Since applications vary, each investigator should titrate the reagent to obtain optimal results.
Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
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.
The WT.5 monoclonal antibody specifically recognizes the alpha subunit of LFA-1 (αLβ2 integrin, CD11a/CD18), a heterodimeric surface glycoprotein which is found on the majority of leukocytes, but not on peritoneal macrophages or peritoneal mast cells. LFA-1 mediates a variety of heterotypic and homotypic intercellular adhesions through interaction with ICAM-1 (CD54) and ICAM-2 (CD102). WT.1 mAb recognizes both the activated and unactivated forms of LFA-1. It inhibits the binding of LFA-1 to ICAM-1 in several in vitro assays, including binding of Concanavalin A-stimulated lymphocytes (Con A blasts) to purified ICAM-1 and Mg2+-dependent aggregation of concanavalin A-stimulated blasts. It has also been reported to inhibit leukocyte infiltration in several in vivo models of inflammation.
This antibody is routinely tested by flow cytometric analysis. Other applications were tested at BD Biosciences Pharmingen during antibody development only or reported in the literature.
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R-Phycoerythrin (PE), is part of the BD family of Phycobiliprotein dyes. This fluorochrome is a multimeric fluorescent phycobiliprotein with excitation maximum (Ex Max) of 496 nm and 566 nm and an emission maximum (Em Max) at 576 nm. PE is designed to be excited by the Blue (488 nm), Green (532 nm) and Yellow-Green (561 nm) lasers and detected using an optical filter centered near 575 nm (e.g., a 575/26-nm bandpass filter). As PE is excited by multiple lasers, this can result in cross-laser excitation and fluorescence spillover on instruments with various combinations of Blue, Green, and Yellow-Green lasers. Please ensure that your instrument’s configurations (lasers and optical filters) are appropriate for this dye.
Yellow-Green 488 nm, 532 nm, 561 nm
496 nm, 566 nm
Citations & References
Bañuls MP, Alvarez A, Ferrero I, Zapata A, Ardavin C. Cell-surface marker analysis of rat thymic dendritic cells. Immunology. 1993; 79(2):298-304. (Clone-specific).
Fox CC, Jewell SD, Whitacre CC. Rat peritoneal mast cells present antigen to a PPD-specific T cell line. Cell Immunol. 1994; 158(1):253-264. (Clone-specific).
Kawasaki K, Yaoita E, Yamamoto T, Tamatani T, Miyasaka M, Kihara I. Antibodies against intercellular adhesion molecule-1 and lymphocyte function-associated antigen-1 prevent glomerular injury in rat experimental crescentic glomerulonephritis. J Immunol. 1993; 150(3):1074-1083. (Clone-specific).
Larson RS, Springer TA. Structure and function of leukocyte integrins. Immunol Rev. 1990; 114:181-217. (Biology).
Nishikawa K, Guo YJ, Miyasaka M, et al. Antibodies to intercellular adhesion molecule 1/lymphocyte function-associated antigen 1 prevent crescent formation in rat autoimmune glomerulonephritis. J Exp Med. 1993; 177(3):667-677. (Clone-specific).
Springer TA. Traffic signals for lymphocyte recirculation and leukocyte emigration: the multistep paradigm. Cell. 1994; 76(2):301-314. (Clone-specific).
Tamatani T, Kotani M, Miyasaka M. Characterization of the rat leukocyte integrin, CD11/CD18, by the use of LFA-1 subunit-specific monoclonal antibodies. Eur J Immunol. 1991; 21(3):627-633. (Immunogen).
Wada J, Shikata K, Makino H, et al. The critical role of intercellular adhesion molecule-1 in Masugi nephritis in rats. Nephron. 1996; 73(2):264-272. (Clone-specific).
Watanabe T, Arakawa T, Fukuda T, Higuchi K, Kobayashi K. Role of neutrophils in a rat model of gastric ulcer recurrence caused by interleukin-1 beta. Am J Pathol. 1997; 150(3):971-979. (Clone-specific).
Yamazaki T, Seko Y, Tamatani T, et al. Expression of intercellular adhesion molecule-1 in rat heart with ischemia/reperfusion and limitation of infarct size by treatment with antibodies against cell adhesion molecules. Am J Pathol. 1993; 143(2):410-418. (Clone-specific).