Biolegend, 313513, Alexa Fluor® 488 anti-human/mouse/rat CD278 (ICOS) Antibody, 25μg

ICOS, also known as inducible costimulatory molecule and H4, is a 47-57 kD protein. This protein is homologous to the CD28/CTLA-4 proteins. ICOS is expressed on activated T cells and a subset of thymocytes. It is able to costimulate T cells proliferation. In addition, ICOS is involved in humoral immune responses (B cell germinal center formation). The ICOS ligand is B7h/B7RP-1 or B7-H2. ICOS stimulation has been shown to potentiate TCR-mediated IL-4 and IL-10 production and has been proposed to play a role in Th2 cell development.
25μg
Verified Reactivity: Human, Mouse, Rat
Reported Reactivity: African Green, Baboon, Cynomolgus, Rhesus, Pig
Antibody Type: Monoclonal
Host Species: Armenian Hamster
Immunogen: Mouse T cell clone D10.G4.1
Formulation: Phosphate-buffered solution, pH 7.2, containing 0.09% sodium azide.
Preparation: The antibody was purified by affinity chromatography and conjugated with Alexa Fluor® 488 under optimal conditions.
Concentration: 0.5 mg/ml
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 - Reported in the literature, not verified in house
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 ≤ 0.5 µg per 106 cells in 100 µl volume. It is recommended that reagents be titrated for optimal performance in the particular application. * Alexa Fluor® 488 has a maximum emission of 519 nm when it is excited at 488 nm. Alexa Fluor® and Pacific Blue™ are trademarks of Life Technologies Corporation.View full statement regarding label licenses
Excitation Laser: Blue Laser (488 nm)
Application Notes: The C398.4A antibody is useful for flow cytometric analysis and is able to costimulate T cell activation and proliferation. Additional reported applications (for the relevant formats) include: immunoprecipitation1, in vitro costimulation of T cell activation1,3,4, and spatial biology (IBEX)5,6. The LEAF™ purified antibody (Endotoxin < 0.1 EU/µg, Azide-Free, 0.2 µm filtered) is recommended for functional assays (Cat. No. 313512).
Additional Product Notes: Iterative Bleaching Extended multi-pleXity (IBEX) is a fluorescent imaging technique capable of highly-multiplexed spatial analysis. The method relies on cyclical bleaching of panels of fluorescent antibodies in order to image and analyze many markers over multiple cycles of staining, imaging, and, bleaching. It is a community-developed open-access method developed by the Center for Advanced Tissue Imaging (CAT-I) in the National Institute of Allergy and Infectious Diseases (NIAID, NIH).
Application References(PubMed link indicates BioLegend citation): Redoglia V, et al. 1996. Eur. J. Immunol. 26:2781. (FC IP Costim) Yagi J, et al. 2003. J. Immunol. 171:783. (FC) Arimura Y, et al. 2002. Int. Immunol. 14:555. (Costim) Arimura Y, et al. 2004. J. Biol. Chem. 279:11408. (Costim) Radtke AJ, et al. 2020. Proc Natl Acad Sci USA. 117:33455-33465. (SB) PubMed Radtke AJ, et al. 2022. Nat Protoc. 17:378-401. (SB) PubMed
Product Citations: Matsui K, et al. 2015. PLoS One. 10: 0137195. PubMed Yue X, et al. 2019. Nat Commun. 10:2011. PubMed Shuwa HA, et al. 2021. Med. 2(6):720-735.e4. PubMed Masuda N, et al. 2021. BMC Cancer. 21:1269. PubMed Lederer K, et al. 2020. Immunity. 53(6):1281-1295.e5. PubMed
RRID: AB_2122584 (BioLegend Cat. No. 313514)
Structure: CD28/CTLA-4, 47-57 kD
Distribution: Activated T cells, subset of thymocytes
Function: Costimulates T cell activation, proliferation, humoral immune response
Ligand/Receptor: B7h/B7RP-1/GL-50
Cell Type: T cells, Thymocytes, Tregs
Biology Area: Costimulatory Molecules, Immunology
Molecular Family: CD Molecules
Antigen References: 1. Redoglia V, et al. 1996. Eur. J. Immunol. 26:2781. 2. Hutloff A, et al. 1999. Nature 397:263. 3. Buonfiglio D, et al. 2000. Eur. J. Immunol. 30:3463. 4. Coyle AJ, et al. 2000. Immunity 13:95.
Gene ID: 1000488412985164545
UniProt: View information about CD278 on UniProt.org
Clone: C398.4A
Regulatory Status: RUO
Other Names: Inducible COStimulatory molecule, H4
Isotype: Armenian Hamster IgG
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.