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Multiparameter flow cytometric analysis of CD54 expression on human peripheral blood leucocytes. Human whole blood was stained with either BD Horizon™ RY586 Mouse IgG1, κ Isotype Control (Cat. No. 568097; Left Plot) or BD Horizon RY586 Mouse Anti-Human CD54 antibody (Cat. No. 568123/568122; Right Plot). Erythrocytes were lysed with BD FACS Lysing Solution (Cat. No. 349202). Two-parameter flow cytometric contour plots showing the correlated expression of CD54 (or Ig Isotype control staining) versus side light-scatter signals were derived from gated events with the forward and side light-scatter characteristics of intact leucocyte populations. Flow cytometric analysis was performed using a BD LSRFortessa™ Flow Cytometer System and FlowJo™ software.
BD Horizon™ RY586 Mouse Anti-Human CD54 (ICAM-1)
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Preparation And Storage
Recommended Assay Procedures
BD® CompBeads can be used as surrogates to assess fluorescence spillover (compensation). When fluorochrome conjugated antibodies are bound to BD® CompBeads, they have spectral properties very similar to cells. However, for some fluorochromes there can be small differences in spectral emissions compared to cells, resulting in spillover values that differ when compared to biological controls. It is strongly recommended that when using a reagent for the first time, users compare the spillover on cells and BD® CompBeads to ensure that BD® CompBeads are appropriate for your specific cellular application.
Product Notices
- Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
- 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.
- 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.
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- Human donor specific background has been observed in relation to the presence of anti-polyethylene glycol (PEG) antibodies, developed as a result of certain vaccines containing PEG, including some COVID-19 vaccines. We recommend use of BD Horizon Brilliant™ Stain Buffer in your experiments to help mitigate potential background. For more information visit https://www.bdbiosciences.com/en-us/support/product-notices.
- For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
Companion Products
The HA58 monoclonal antibody specifically binds to CD54 which is also known as ICAM-1 (intracellular adhesion molecule-1). CD54 is an 85-110 kDa type I transmembrane glycoprotein that belongs to the immunoglobulin supergene family. A soluble form of CD54 can also be found in biological fluids. CD54 is expressed on endothelial cells and both resting (weak) and activated (moderate) lymphocytes and monocytes. CD54 is a ligand for LFA-1 (CD11a/CD18) and Mac-1 (CD11b/CD18). The CD54 adhesion molecule plays roles in inflammatory and immune responses and neoplasia. This antibody (NA/LE format) blocks the mixed-lymphocyte reaction (MLR) and the purified format is suitable for staining acetone-fixed, frozen tissue sections.
The BD Horizon RealYellow™ 586 (RY586) Dye is part of the BD family of yellow-green dyes. It is a small organic fluorochrome with an excitation maximum (Ex Max) at 565-nm and an emission maximum (Em Max) at 586-nm. Driven by BD innovation, RY586 can be used on both spectral and conventional cytometers and is designed to be excited by the Yellow-Green laser (561-nm) with minimal excitation by the 488-nm Blue laser. For conventional instruments equipped with a Yellow-Green laser (561-nm), RY586 can be used as an alternative to PE and we recommend using an optical filter centered near 586-nm (eg, a 586/15-nm bandpass filter). For spectral instruments equipped with a Yellow-Green laser (561-nm), it can be used in conjunction with PE. Compared to PE, RY586 is similar in brightness, minimal spillover into Blue detectors, and increased spillover into the 610/20-nm (PE-CF594) detector. Please ensure that your instrument configuration (lasers and optical filters) is appropriate for this dye.
Development References (5)
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Imai K, Tsujisaki M, Hareyama M, Hinoda Y. CD54 Workshop Panel Report. In: Kishimoto T. Tadamitsu Kishimoto .. et al, ed. Leucocyte typing VI : white cell differentiation antigens : proceedings of the sixth international workshop and conference held in Kobe, Japan, 10-14 November 1996. New York: Garland Pub.; 1997:409-411.
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Kishimoto T. Tadamitsu Kishimoto .. et al., ed. Leucocyte typing VI : white cell differentiation antigens : proceedings of the sixth international workshop and conference held in Kobe, Japan, 10-14 November 1996. New York: Garland Pub.; 1997.
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Knapp W. W. Knapp .. et al., ed. Leucocyte typing IV : white cell differentiation antigens. Oxford New York: Oxford University Press; 1989:1-1182.
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Schlossman SF. Stuart F. Schlossman .. et al., ed. Leucocyte typing V : white cell differentiation antigens : proceedings of the fifth international workshop and conference held in Boston, USA, 3-7 November, 1993. Oxford: Oxford University Press; 1995.
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Tsujisaki M, Imai K, Hirata H, et al. Detection of circulating intercellular adhesion molecule-1 antigen in malignant diseases. Clin Exp Immunol. 1991; 85(1):3-8. (Immunogen: Blocking, ELISA, Flow cytometry, Immunoprecipitation, Radioimmunoassay, Western blot). View Reference
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For Research Use Only. Not for use in diagnostic or therapeutic procedures.