Spark YG™ 581 anti-human CD3 (Flexi-Fluor™) Antibody

Product Details

Verified Reactivity

Human

Antibody Type

Monoclonal

Host Species

Mouse

Formulation

Phosphate-buffered solution, pH 7.2, containing 0.09% sodium azide

Preparation

The antibody was purified by affinity chromatography and conjugated with Spark YG™ 581 under optimal conditions.

Concentration

0.2 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

Recommended Usage

Flexi-Fluors™ are provided at a standard 0.2 mg/mL concentration. We recommend titrating this reagent to determine the optimal concentration for each application. For many flow cytometry applications, conjugated antibodies perform well at concentrations ranging from 0.03 to 1.0 µg per million cells in 100 µL. We recommend testing a range of concentrations starting from 10 µg/mL.

For example, make five 1:1 serial dilutions of the 0.2 mg/mL antibody. Add 5 µL of each dilution (including the undiluted antibody) to 100 µL of cells (at 10⁷ cells/mL) to test six concentrations -- 1.0, 0.5, 0.25, 0.125, 0.06, and 0.03 µg per million cells in 100 µL volume. Compare staining patterns or create a titration curve using the MFI or staining index to determine the optimal concentration.

* Spark YG™ 581 has a maximum excitation of 562 nm and a maximum emission of 581 nm.

Excitation Laser

Green Laser (532 nm)/Yellow-Green Laser (561 nm)

Application Notes

Additional reported (for the relevant formats) applications include: immunohistochemical staining of acetone-fixed frozen sections, immunoprecipitation, and activation of T lymphocytes^4-7. The HIT3a antibody is able to stimulate T cell activation. The LEAF™ purified antibody (Endotoxin <0.1 EU/µg, Azide-Free, 0.2 µm filtered) is recommended for functional assays (Cat. No. 300314). For highly sensitive assays, we recommend Ultra-LEAF™ purified antibody (Cat. No. 300332) with a lower endotoxin limit than standard LEAF™ purified antibodies (Endotoxin <0.01 EU/µg).

Additional Product Notes

For more information about Flexi-Fluors™, visit our Flexi-Fluor™ page and review FAQs associated with this product line.

Application References

1. Schlossman S, et al. Eds. 1995. Leucocyte Typing V. Oxford University Press. New York.
2. Knapp W. 1989. Leucocyte Typing IV. Oxford University Press New York.
3. Barclay N, et al. 1997. The Leucocyte Antigen Facts Book. Academic Press Inc. San Diego.
4. Sedelies KA, et al. 2004. J. Biol. Chem. 279:26581. (Activ)
5. Rivollier A, et al. 2004. Blood 104:4029. (Activ)
6. Scharschmidt E, et al. 2004. Mol. Cell Biol. 24:3860. (Activ)
7. Smeltz RB. 2007. J. Immunol. 178:4786. (Activ)

RRID

AB_3717029 (BioLegend Cat. No. 285398)

Antigen Details

Structure

Ig superfamily, with the subunits of CD3γ, CD3δ, CD3ζ (CD247) and TCR (α/β or γ/δ) forms CD3/TCR complex, 20 kD

Distribution

Mature T and NK-T cells, thymocyte differentiation

Function

Antigen recognition, signal transduction, T cell activation

Ligand/Receptor

Peptide antigen bound to MHC

Cell Type

NKT cells, T cells, Thymocytes, Tregs

Biology Area

Immunology

Molecular Family

CD Molecules, TCRs

Antigen References

1. Barclay N, et al. 1993. The Leucocyte FactsBook. Academic Press. San Diego.
2. Beverly P, et al. 1981. Eur. J. Immunol. 11:329.
3. Lanier L, et al. 1986. J. Immunol. 137:2501-2507.

Gene ID

NA

UniProt

View information about CD3 on UniProt.org

Documentation

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

Reviews

Related Protocols

• Cell Surface Flow Cytometry Staining Protocol

Related Pages & Pathways

Related FAQs

What are Flexi-Fluors?

Flexi-Fluors are rapidly made-to-order conjugated antibodies. The technology, manufacturing processes, and specifications used to create Flexi-Fluors are the same as our regular catalog products. However, the optimal concentration and performance of each Flexi-Fluor must be determined by the customer. 

How quickly will I receive my order?

We aim to ship Flexi-Fluors within 2-3 weeks of receipt of your order. However, depending on your location, shipping times may vary. 

How are Flexi-Fluors different from regular catalog products?

Flexi-Fluors are made on demand, specifically for you. Flexi-Fluors are manufactured using the same high-quality standards, and specifications as other catalog products. For faster delivery, Flexi-Fluors are not tested by flow cytometry to determine optimal concentrations or evaluate performance. This testing needs to be performed by the customer.

How do I determine the optimal concentration for using my Flexi-Fluor? How should I titrate my antibody?

Flexi-Fluors are provided at a standard 0.2 mg/mL concentration. We recommend that you titrate your antibody to determine the optimal concentration to use for your application. For many flow cytometry applications, conjugated antibodies perform well at concentrations ranging from 0.03 to 1.0 µg per million cells in 100 µL volume. We recommend that you test a range of concentrations starting from 10 µg/mL. 

 

For example, make five 1:1 serial dilutions of your 0.2 mg/mL antibody.  Add 5 µL of each dilution (including the undiluted antibody) to 100 µL of cells (at 10⁷ cells/ml) to test six concentrations - 1.0, 0.5, 0.25, 0.125, 0.06, and 0.03 µg per million cells in 100 µL volume. Compare staining patterns or create a titration curve using the MFI or staining index to determine the optimal concentration. 

I can’t find the antibody-dye combination that I need. When will it be available?

We continuously update our catalog, introducing scores of new products every month. Please get in touch with our Technical Service team for an update on new products or recommendations for suitable alternatives to complete your panel. Or contact Custom Solutions to inquire about our affordable custom conjugation services. 

I need help to validate the performance of my Flexi-Fluor. Who should I contact?

Please get in touch with Technical Service for assistance. 

Can I order more than 50 μg of a Flexi-Fluor?

Yes, you can order multiple vials of the same Flexi-Fluor products.  We cannot guarantee, however, that these vials will be bottled from the same lot. For bulk single-lot orders, contact our Custom Solutions team.

What is the expiration date of my Flexi-Fluor?

Expiration dates can be found on the vial label or by using our CoA lookup tool. 

What if my requested antibody-dye combination doesn't perform as expected?

Flexi-Fluor™ products have been carefully selected by our scientists for their excellent conjugation properties and broad compatibility. In the rare case that an antibody-dye combination does not perform as expected due to the unique characteristics of a specific pairing, please contact our Technical Service team. We will work with you to identify alternative options to meet your panel requirements.

Other Formats

Certificate of Analysis