Biolegend, 306006, PE anti-human CD123 Antibody, 100tests

CD123 is the 70 kD transmembrane α chain of the IL-3 receptor. Alone, CD123 binds IL-3 with low affinity; when CD123 associates with CD131 (common β chain), it binds IL-3 with high affinity. CD123 does not transduce intracellular signals upon binding IL-3 and requires the β chain for this function. CD123 is expressed by myeloid precursors, macrophages, dendritic cells, mast cells, basophils, megakaryocytes, and some B cells.
100tests
Verified Reactivity: Human
Reported Reactivity: Rhesus
Antibody Type: Monoclonal
Host Species: Mouse
Immunogen: Human IL-3Rα transfected COS cells.
Formulation: Phosphate-buffered solution, pH 7.2, containing 0.09% sodium azide and BSA (origin USA)
Preparation: The antibody was purified by affinity chromatography, and conjugated with PE under optimal conditions.
Concentration: Lot-specific (to obtain lot-specific concentration and expiration, please enter the lot number in our Certificate of Analysis online tool.)
Storage & Handling: The antibody solution should be stored undiluted between 2°C and 8°C, and protected from prolonged exposure to light. Do not freeze.
Application: FC - Quality tested SB - Community Verified
Recommended Usage: Each lot of this antibody is quality control tested by immunofluorescent staining with flow cytometric analysis. For flow cytometric staining, the suggested use of this reagent is 5 µl per million cells in 100 µl staining volume or 5 µl per 100 µl of whole blood.
Excitation Laser: Blue Laser (488 nm)Green Laser (532 nm)/Yellow-Green Laser (561 nm)
Application Notes: Clone 6H6 does not inhibit IL-3 binding to low- or high-affinity IL-3Rs. Additional reported applications (for the relevant formats) include: Western blotting1, immunoprecipitation1, and immunohistochemical staining of acetone-fixed frozen sections2 and also paraformaldehyde fixed paraffin embedded tissue7.
Additional Product Notes: For the use of this antibody in spatial biology (SB), we have partnered with Bruker Spatial Biology Biosciences for demonstration of this antibody on their next-generation ChipCytometry instrument called the CellScape™. The CellScape platform is an end-to-end solution for highly multiplexed spatial omics. Combining an advanced, purpose-built imaging system with easy-to-use fluidics for walk-away automation, the CellScape system will accelerate your exploration into the rapidly evolving field of spatial biology. More information on the the Bruker Spatial Biology CellScape and a complete list of our antibodies that have been demonstrated on the instrument can be found here.
Application References(PubMed link indicates BioLegend citation): Sun Q, et al. 1996. Blood 87:83. (IP, WB) Herling M, et al. 2003. Blood 101:5007. (IHC) Charles N, et al. 2010. Nat. Med. 16:701. (FC) PubMed Martin-Gayo E, et al. 2010. Blood 115:5366. PubMed Chen SC, et al. 2010. Arch Dermatol Res. 302:113. PubMed Liu Y, et al. 2012. Food Chem Toxicol. 50:1920. PubMed Peduzzi E, et al. 2007. J. Invest. Dermatol. 127:638. (IHC)
Product Citations: Woo MS, et al. 2021. iScience. 24(7):102752. PubMed Zaro BW, et al. 2019. J Immunol. 202:2737. PubMed Luchsinger LL, et al. 2020. Cell Stem Cell. 25(2):225-240. PubMed Randrianarison-Huetz V, et al. 2010. Blood. 115:2784. PubMed Li M, et al. 2023. Front Immunol. 14:1087923. PubMed Sefik E, et al. 2022. Nature. 606:585. PubMed Shahid AM, et al. 2022. PLoS One. 17:e0278209. PubMed Baudet A, et al. 2022. Bio Protoc. 12:e4353. PubMed Han L, et al. 2012. PLoS One. 4:e7989. PubMed Jarosch S, et al. 2022. STAR Protoc. 3:101374. PubMed Kim ST, et al. 2022. Nat Commun. 13:1970. PubMed Jarosch S, et al. 2021. Cell Rep Methods. 1:100104. PubMed Warmuth S, et al. 2022. Oncoimmunology. 10:2004661. PubMed Sefik E, et al. 2021. Nat Biotechnol. . PubMed Houtsma R, et al. 2021. STAR Protoc. 2:100864. PubMed Le J, et al. 2020. Immunity. 52(6):1105-1118.e9. PubMed Felce JH, et al. 2018. Sci Signal. 11:eaat0756. PubMed Li M, et al. 2020. Nat Commun. 4051:11. PubMed Kerdidani D, et al. 2022. J Exp Med. 219:. PubMed Leylek R, et al. 2020. Cell Rep. 32:108180. PubMed
RRID: AB_314579 (BioLegend Cat. No. 306005) AB_314580 (BioLegend Cat. No. 306006)
Structure: Ig superfamily, type I transmembrane glycoprotein, associates with CDw131, 70 kD
Distribution: Myeloid precursors, basophils, mast cells, macrophages, dendritic cells, megakaryocytes, subset of lymphocytes
Function: Hematopoietic cell proliferation, differentiation
Ligand/Receptor: IL-3
Cell Type: Basophils, Dendritic cells, Hematopoietic stem and progenitors, Lymphocytes, Macrophages, Mast cells, Megakaryocytes
Biology Area: Immunology
Molecular Family: CD Molecules, Cytokine/Chemokine Receptors
Antigen References: 1. Miyajima A, et al. 1993. Blood 82:1960.
Gene ID: 3563
UniProt: View information about CD123 on UniProt.org
Clone: 6H6
Regulatory Status: RUO
Other Names: IL-3Rα, IL-3 Receptor alpha
Isotype: Mouse IgG1, κ
Q: What type of PE do you use in your conjugates?
A: We use R-PE in our conjugates.
Q: If an antibody clone has been previously successfully used in IBEX in one fluorescent format, will other antibody formats work as well?
A: It’s likely that other fluorophore conjugates to the same antibody clone will also be compatible with IBEX using the same sample fixation procedure. Ultimately a directly conjugated antibody’s utility in fluorescent imaging and IBEX may be specific to the sample and microscope being used in the experiment. Some antibody clone conjugates may perform better than others due to performance differences in non-specific binding, fluorophore brightness, and other biochemical properties unique to that conjugate.
Q: Will antibodies my lab is already using for fluorescent or chromogenic IHC work in IBEX?
A: Fundamentally, IBEX as a technique that works much in the same way as single antibody panels or single marker IF/IHC. If you’re already successfully using an antibody clone on a sample of interest, it is likely that clone will have utility in IBEX. It is expected some optimization and testing of different antibody fluorophore conjugates will be required to find a suitable format; however, legacy microscopy techniques like chromogenic IHC on fixed or frozen tissue is an excellent place to start looking for useful antibodies.
Q: Are other fluorophores compatible with IBEX?
A: Over 18 fluorescent formats have been screened for use in IBEX, however, it is likely that other fluorophores are able to be rapidly bleached in IBEX. If a fluorophore format is already suitable for your imaging platform it can be tested for compatibility in IBEX.
Q: The same antibody works in one tissue type but not another. What is happening?
A: Differences in tissue properties may impact both the ability of an antibody to bind its target specifically and impact the ability of a specific fluorophore conjugate to overcome the background fluorescent signal in a given tissue. Secondary stains, as well as testing multiple fluorescent conjugates of the same clone, may help to troubleshoot challenging targets or tissues. Using a reference control tissue may also give confidence in the specificity of your staining.
Q: How can I be sure the staining I’m seeing in my tissue is real?
A: In general, best practices for validating an antibody in traditional chromogenic or fluorescent IHC are applicable to IBEX. Please reference the Nature Methods review on antibody based multiplexed imaging for resources on validating antibodies for IBEX.