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92511
Human CD3ε Activating (OKT3) Mouse mAb (Low Endotoxin, Azide-free)
Primary Antibodies
Monoclonal Antibody

Human CD3ε Activating (OKT3) Mouse mAb (Low Endotoxin, Azide-free) #92511

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Human CD3ε Activating (OKT3) Mouse mAb (Low Endotoxin, Azide-free): Image 1
Flow cytometric analysis of Jurkat cells, untreated (blue) or treated with cross-linked Human CD3ε Activating (OKT3) Mouse mAb (Low Endotoxin, Azide-free) and Human CD28 Activating (CD28.2) Mouse mAb (Low Endotoxin, Azide-free) #91920 (10 μg/ml each, 15 min; green), using Phospho-SLP-76 (Ser376) (E3G9U) XP® Rabbit mAb #76384 (solid lines) or concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (dashed lines). Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.
Human CD3ε Activating (OKT3) Mouse mAb (Low Endotoxin, Azide-free): Image 2
Western blot analysis of extracts from Jurkat cells, untreated (1) or cross-linked using Human CD3ε Activating (OKT3) Mouse mAb (Low Endotoxin, Azide-free) and Human CD28 Activating (CD28.2) Mouse mAb (Low Endotoxin, Azide-free) #91920 (10 μg/ml, 30 min) (2), using Phospho-SLP-76 (Ser376) (D7S1K) Rabbit mAb #92711 (upper), SLP-76 (E4N7E) Rabbit mAb #25361 (middle), and β-Actin (D6A8) Rabbit mAb #8457 (lower).
To Purchase # 92511
Cat. # Size Qty. Price
92511S
100 µg $ 149

Supporting Data

REACTIVITY
SENSITIVITY Endogenous
MW (kDa)
Source/Isotype Mouse IgG2a kappa

Application Key:

  • WB-Western Blot
  • IP-Immunoprecipitation
  • IHC-Immunohistochemistry
  • ChIP-Chromatin Immunoprecipitation
  • IF-Immunofluorescence
  • F-Flow Cytometry
  • E-P-ELISA-Peptide

Species Cross-Reactivity Key:

  • H-Human
  • M-Mouse
  • R-Rat
  • Hm-Hamster
  • Mk-Monkey
  • Vir-Virus
  • Mi-Mink
  • C-Chicken
  • Dm-D. melanogaster
  • X-Xenopus
  • Z-Zebrafish
  • B-Bovine
  • Dg-Dog
  • Pg-Pig
  • Sc-S. cerevisiae
  • Ce-C. elegans
  • Hr-Horse
  • All-All Species Expected

Product Description

Endotoxin Less than or equal to 0.01 EU/μg, as determined by the LaL assay.

Storage

Supplied in 10 mM NaH2PO4, 150 mM NaCl, pH7.2. Stable for 12 months when stored at 4°C. Do not aliquot the antibody.

Protocol

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A. Solutions and Reagents

  1. Human CD3ε Activating (OKT3) Mouse mAb (Low Endotoxin, Azide-free) #92511
  2. Human CD28 Activating (CD28.2) Mouse mAb (Low Endotoxin, Azide-free) #91920
  3. For Soluble Stimulation Only: Goat Anti-Mouse Kappa Light Chain, F(ab’)2 Antibody (Low Endotoxin, Azide-free) #75861

B. Stimulation Protocol: Soluble

  1. Collect cells of interest by centrifugation.
  2. Aspirate the supernatant.
  3. Resuspend cells at 106-107 cells/ml with cold serum-free media.
  4. Add CD3ε Activating (OKT3) Mouse mAb (Low Endotoxin, Azide-free) #92511 to cells at 1:200 to reach a final concentration of 10 µg/ml.
  5. Optional: Add Human CD28 Activating (CD28.2) Mouse mAb (Low Endotoxin, Azide-free) #91920 to cells at 1:200 to reach a final concentration of 10 µg/ml.
  6. Keep cells with agonist antibody on ice for 30 minutes.
  7. Collect cells by centrifugation.
  8. Aspirate the supernatant.
  9. Resuspend cells at 106-107 cells/ml with cold serum-free media and keep on ice.
  10. Add Goat Anti-Mouse Kappa Light Chain, F(ab’)2 Antibody (Low Endotoxin, Azide-free) #75861 secondary antibody to cells at 1:50 to reach a final concentration of 10 µg/ml if only CD3ε (OKT3) was used; add at 1:25 to reach a final concentration of 20 µg/ml if both CD3ε (OKT3) and CD28 (CD28.2) were used.
  11. Keep cells with secondary antibody on ice for 15 minutes.
  12. To initiate stimulation, transfer cells to 37 °C water bath for desired stimulation time.
  13. To terminate stimulation, follow the cell preparation protocol recommended for the desired analysis method.

C. Stimulation Protocol: Plate-based

  1. Prepare a 10 µg/ml solution of CD3ε Activating (OKT3) Mouse mAb (Low Endotoxin, Azide-free) #92511 in sterile PBS. Prepare a sufficient volume to cover the bottom of all the wells to be used for the stimulated T cell condition (approximately 50 µl for 96-well plates, 1 ml for 6-well plates).
  2. Add the diluted CD3ε (OKT3) antibody solution to the wells chosen for treatment. Add equivalent volume of sterile PBS to the wells chosen as controls for treatment.
  3. Cover the plate and incubate at 37 °C for a minimum of 2 hrs. The plate may be allowed to incubate overnight if desired.
  4. Prepare cells as needed to generate a cell suspension of roughly 1 x 106 cells/ml in a sufficient volume of complete media (100 µl per well for 96-well plates, 2 ml for 6-well plates). Optimal cell density may be determined through experimentation with cells of interest. For T cell expansion, add Human Interleukin-2 (hIL-2) #8907 to the cell suspension to yield a final concentration of 20 ng/ml.
  5. Aspirate the CD3ε (OKT3) antibody solution from the wells, and rinse wells 2x with excess sterile PBS to remove unbound antibody.
  6. Add cell suspension to all wells, at volumes specified in step 4 or as determined by experimentation.
  7. Add Human CD28 Activating (CD28.2) Mouse mAb (Low Endotoxin, Azide-free) #91920 to yield a concentration of 2 µg/ml. Pre-dilute CD28 (CD28.2) antibody in complete media if needed. CD28 antibody may also be added to a portion of control wells if desired.
  8. Incubate cells for desired time in a humidified incubator (37 °C, 5% CO2). Add fresh media with IL-2 as needed for T cell expansion.
  9. Harvest cells and proceed with immunostaining or analysis as desired.

Protocol Id: 2450

Specificity / Sensitivity

Human CD3ε Activating (OKT3) Mouse mAb (Low Endotoxin, Azide-free) recognizes endogenous levels of CD3ε protein.

Source / Purification

Monoclonal antibody is produced against human PBMC cells and purified via affinity chromatography.

Background

When T cells encounter antigens via the T cell receptor (TCR), information about the quantity and quality of antigens is relayed to the intracellular signal transduction machinery (1). This activation process depends mainly on CD3 (Cluster of Differentiation 3), a multiunit protein complex that directly associates with the TCR. CD3 is composed of four polypeptides: ζ, γ, ε, and δ. Each of these polypeptides contains at least one immunoreceptor tyrosine-based activation motif (ITAM) (2). Engagement of the TCR complex with foreign antigens induces tyrosine phosphorylation in the ITAM motifs and phosphorylated ITAMs function as docking sites for signaling molecules such as ZAP-70 and the p85 subunit of PI-3 kinase (3,4). TCR ligation also induces a conformational change in CD3ε, such that a proline region is exposed and then associates with the adaptor protein Nck (5).
  1. Kuhns, M.S. et al. (2006) Immunity 24, 133-139.
  2. Pitcher, L.A. and van Oers, N.S. (2003) Trends Immunol. 24, 554-560.
  3. Osman, N. et al. (1996) Eur. J. Immunol. 26, 1063-1068.
  4. Hatada, M.H. et al. (1995) Nature 377, 32-38.
  5. Gil, D. et al. (2002) Cell 109, 901-912.

Pathways & Proteins

Explore pathways + proteins related to this product.

Limited Uses

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Products are labeled with For Research Use Only or a similar labeling statement and have not been approved, cleared, or licensed by the FDA or other regulatory foreign or domestic entity, for any purpose. Customer shall not use any Product for any diagnostic or therapeutic purpose, or otherwise in any manner that conflicts with its labeling statement. Products sold or licensed by CST are provided for Customer as the end-user and solely for research and development uses. Any use of Product for diagnostic, prophylactic or therapeutic purposes, or any purchase of Product for resale (alone or as a component) or other commercial purpose, requires a separate license from CST. Customer shall (a) not sell, license, loan, donate or otherwise transfer or make available any Product to any third party, whether alone or in combination with other materials, or use the Products to manufacture any commercial products, (b) not copy, modify, reverse engineer, decompile, disassemble or otherwise attempt to discover the underlying structure or technology of the Products, or use the Products for the purpose of developing any products or services that would compete with CST's products or services, (c) not alter or remove from the Products any trademarks, trade names, logos, patent or copyright notices or markings, (d) use the Products solely in accordance with CST's Product Terms of Sale and any applicable documentation, and (e) comply with any license, terms of service or similar agreement with respect to any third party products or services used by Customer in connection with the Products.

For Research Use Only. Not For Use In Diagnostic Procedures.
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