Alexa Fluor® 647 anti-human CD4 Antibody

Product Details

Verified Reactivity

Human

Reported Reactivity

Chimpanzee

Antibody Type

Monoclonal

Host Species

Mouse

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 Alexa Fluor® 647 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 - 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 5 µl per million cells in 100 µl staining volume or 5 µl per 100 µl of whole blood.

* Alexa Fluor® 647 has a maximum emission of 668 nm when it is excited at 633nm / 635nm.

Alexa Fluor® and Pacific Blue™ are trademarks of Life Technologies Corporation.

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Excitation Laser

Red Laser (633 nm)

Application Notes

The RPA-T4 antibody binds to the D1 domain of CD4 (CDR1 and CDR3 epitopes) and can block HIV gp120 binding and inhibit syncytia formation. Additional reported applications (for the relevant formats) include: immunohistochemistry of acetone-fixed frozen sections^3,4,5, blocking of T cell activation^1,2, and spatial biology (IBEX)^10,11.  This clone was tested in-house and does not work on formalin fixed paraffin-embedded (FFPE) tissue. The Ultra-LEAF™ purified antibody (Endotoxin < 0.01 EU/µg, Azide-Free, 0.2 µm filtered) is recommended for functional assays (Cat. No. 300569 - 300574).

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)

1. Knapp W, et al. 1989. Leucocyte Typing IV. Oxford University Press. New York. (Activ)
2. Moir S, et al. 1999. J. Virol. 73:7972. (Activ)
3. Deng MC, et al. 1995. Circulation 91:1647. (IHC)
4. Friedman T, et al. 1999. J. Immunol. 162:5256. (IHC)
5. Mack CL, et al. 2004. Pediatr. Res. 56:79. (IHC)
6. Lan RY, et al. 2006. Hepatology 43:729.
7. Zenaro E, et al. 2009. J. Leukoc. Biol. 86:1393. (FC) PubMed
8. Yoshino N, et al. 2000. Exp. Anim. (Tokyo) 49:97. (FC)
9. Stoeckius M, et al. 2017. Nat. Methods. 14:865. (PG)
10. Radtke AJ, et al. 2020. Proc Natl Acad Sci USA. 117:33455-33465. (SB) PubMed
11. Radtke AJ, et al. 2022. Nat Protoc. 17:378-401. (SB) PubMed

Product Citations

1. Junker F, et al. 2021. Cytometry A. 99:832. PubMed
2. Lu T, et al. 2016. PLoS One. 11: 0148044. PubMed
3. NULL, et al. 2020. J Clin Immunol. 40:1. PubMed
4. Riese P, et al. 2022. Nat Commun. 13:6894. PubMed
5. O'Connor MH, et al. 2021. Commun Biol. 4:563. PubMed
6. Parent AV, et al. 2021. Cell Reports. 36(7):109538. PubMed

RRID

AB_389333 (BioLegend Cat. No. 300523)
AB_389333 (BioLegend Cat. No. 300520)

Antigen Details

Structure

Ig superfamily, type I transmembrane glycoprotein, 55 kD

Distribution

T cell subset, majority of thymocytes, monocytes/macrophages

Function

MHC class II co-receptor, lymphocyte adhesion, thymic differentiation, HIV receptor

Ligand/Receptor

MHC class II molecules, HIV gp120, IL-16

Cell Type

Dendritic cells, Macrophages, Monocytes, T cells, Thymocytes, Tregs

Biology Area

Immunology

Molecular Family

CD Molecules

Antigen References

1. Center D, et al. 1996. Immunol. Today 17:476.
2. Gaubin M, et al. 1996. Eur. J. Clin. Chem. Clin. Biochem. 34:723.

Gene ID

920 View all products for this Gene ID

Specificity (DOES NOT SHOW ON TDS):

CD4

Specificity Alt (DOES NOT SHOW ON TDS):

CD4

App Abbreviation (DOES NOT SHOW ON TDS):

FC,SB

UniProt

View information about CD4 on UniProt.org

Documentation

• Technical Data Sheet (pdf)
• Certificate of Analysis
• Safety Data Sheet

Reviews

Related Protocols

• Cell Surface Flow Cytometry Staining Protocol

Related Pages & Pathways

Related FAQs

I am unable to see expression of T cell markers such as CD3 and CD4 post activation.

TCR-CD3 complexes on the T-lymphocyte surface are rapidly downregulated upon activation with peptide-MHC complex, superantigen or cross-linking with anti-TCR or anti-CD3 antibodies. PMA/Ionomycin treatment has been shown to downregulate surface CD4 expression. Receptor downregulation is a common biological phenomenon and so make sure that your stimulation treatment is not causing it in your sample type.

If an antibody clone has been previously successfully used in IBEX in one fluorescent format, will other antibody formats work as well?

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.

Will antibodies my lab is already using for fluorescent or chromogenic IHC work in IBEX?

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.

Are other fluorophores compatible with IBEX?

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.

The same antibody works in one tissue type but not another. What is happening?

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.

How can I be sure the staining I’m seeing in my tissue is real?

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.

Other Formats

Certificate of Analysis