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15135
OX40 (D1S6L) Rabbit mAb (Alexa Fluor® 488 Conjugate)
Antibody Conjugates

OX40 (D1S6L) Rabbit mAb (Alexa Fluor® 488 Conjugate) #15135

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  1. IF
  2. F

Confocal immunofluorescent analysis of HuT 102 (left) and Jurkat (right) cells using OX40 (D1S6L) Rabbit mAb (Alexa Fluor® 488 Conjugate) (green). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).

Flow cytometric analysis of human peripheral blood mononuclear cells, untreated (left) or PHA-treated (1 μg/ml, 48 hr, 37ºC; right), using OX40 (D1S6L) Rabbit mAb (Alexa Fluor® 488 Conjugate) and co-stained with an anti-human CD4 antibody.

To Purchase # 15135S
Product # Size Price
15135S
100 µl  (50 tests) $ 210

Supporting Data

REACTIVITY H
SENSITIVITY Endogenous
MW (kDa)
Isotype Rabbit IgG

Application Key:

  • W-Western
  • 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
  • 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

This Cell Signaling Technology antibody is conjugated to Alexa Fluor® 488 fluorescent dye and tested in-house for direct flow cytometry analysis in human cells. This antibody is expected to exhibit the same species cross-reactivity as the unconjugated OX40 (D1S6L) Rabbit mAb #15123.

Product Usage Information

Application Dilution
Immunofluorescence (Immunocytochemistry) 1:50
Flow Cytometry 1:50

Storage:

Supplied in PBS (pH 7.2), less than 0.1% sodium azide and 2 mg/ml BSA. Store at 4°C. Do not aliquot the antibody. Protect from light. Do not freeze.

Protocol

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Immunofluorescence (Immunocytochemistry)

A. Solutions and Reagents

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.

  1. 20X Phosphate Buffered Saline (PBS): (9808) To prepare 1L 1X PBS: add 50 ml 20X PBS to 950 ml dH2O, mix. Adjust pH to 8.0.
  2. Formaldehyde: 16%, methanol free, Polysciences, Inc. (cat# 18814), use fresh and store opened vials at 4°C in dark, dilute in 1X PBS for use.
  3. Blocking Buffer (1X PBS / 5% normal goat serum (#5425) / 0.3% Triton™ X-100): To prepare 10 ml: add 0.5 ml normal goat serum and 0.5 ml 20X PBS to 9.0 ml dH2O, mix. While stirring, add 30 µl Triton™ X-100.
  4. Antibody Dilution Buffer: (1X PBS / 1% BSA / 0.3% Triton™ X-100): To prepare 10 ml, add 30 µl Triton™ X-100 to 10 ml 1X PBS. Mix well then add 0.1 g BSA (#9998), mix.
  5. Prolong® Gold AntiFade Reagent (#9071), Prolong® Gold AntiFade Reagent with DAPI (#8961).

B. Specimen Preparation - Cultured Cell Lines (IF-IC)

NOTE: Cells should be grown, treated, fixed and stained directly in multi-well plates, chamber slides or on coverslips.

  1. Aspirate liquid, then cover cells to a depth of 2–3 mm with 4% formaldehyde diluted in 1X PBS.

    NOTE: Formaldehyde is toxic, use only in a fume hood.

  2. Allow cells to fix for 15 min at room temperature.
  3. Aspirate fixative, rinse three times in 1X PBS for 5 min each.
  4. Proceed with Immunostaining (Section C).

C. Immunostaining

NOTE: All subsequent incubations should be carried out at room temperature unless otherwise noted in a humid light-tight box or covered dish/plate to prevent drying and fluorochrome fading.

  1. Block specimen in Blocking Buffer for 60 min.
  2. While blocking, prepare primary antibody by diluting as indicated on datasheet in Antibody Dilution Buffer.
  3. Aspirate blocking solution, apply diluted primary antibody.
  4. Incubate overnight at 4°C.
  5. Rinse three times in 1X PBS for 5 min each.
  6. Coverslip slides with Prolong® Gold Antifade Reagent (#9071) or Prolong® Gold Antifade Reagent with DAPI (#8961).
  7. For best results, allow mountant to cure overnight at room temperature. For long-term storage, store slides flat at 4°C protected from light.

posted November 2006

revised November 2013

Protocol Id: 182

Flow Cytometry Triton™ X-100 Permeabilization Protocol for Directly Conjugated Antibodies

A. Solutions and Reagents

All reagents required for this protocol may be efficiently purchased together in our Intracellular Flow Cytometry Kit (Triton™ X-100) #51995, or individually using the catalog numbers listed below.

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.

  1. 1X Phosphate Buffered Saline (PBS): To prepare 1 L 1X PBS: add 100 ml 10X PBS (#12528) to 900 ml water, mix.
  2. 4% Formaldehyde, Methanol-Free (#47746)
  3. Cell Permeabilization Buffer: Purchase ready-to-use (#39487) or to prepare 10 ml, add 30 µl Triton™ X-100 to 10 ml Antibody Dilution Buffer. Store at 4°C.
  4. Antibody Dilution Buffer: Purchase ready-to-use Flow Cytometry Antibody Dilution Buffer (#13616), or to prepare 100 ml dissolve 0.5 g Bovine Serum Albumin (BSA) (#9998) in 100 ml 1X PBS. Store at 4°C.

NOTE: When including fluorescent cellular dyes in your experiment (including viability dyes, DNA dyes, etc.), please refer to the dye product page for the recommended protocol. Visit www.cellsignal.com/flowdyes for a listing of cellular dyes validated for use in flow cytometry.

B. Fixation and Permeabilization

NOTE: Adherent cells or tissue should be dissociated and in single-cell suspension prior to fixation.

NOTE: Optimal centrifugation conditions will vary depending upon cell type and reagent volume. Generally, 150-300g for 1-5 minutes will be sufficient to pellet the cells.

NOTE: If using whole blood, lyse red blood cells and wash by centrifugation prior to fixation.

NOTE: Antibodies targeting CD markers or other extracellular proteins may be added prior to fixation if the epitope is disrupted by formaldehyde and/or Triton™ X-100. The antibodies will remain bound to the target of interest during the fixation and permeabilization process. Conduct a small-scale experiment if you are unsure.

  1. Pellet cells by centrifugation and remove supernatant.
  2. Resuspend cells in approximately 100 µl 4% formaldehyde per 1 million cells. Mix well to dissociate pellet and prevent cross-linking of individual cells.
  3. Fix for 15 min at room temperature (20-25°C).
  4. Wash by centrifugation with excess 1X PBS. Discard supernatant in appropriate waste container.
  5. Resuspend cells in approximately 100 µl Cell Permeabilization Buffer per million cells.
  6. Incubate for 10 minutes at room temperature.
  7. Proceed with staining or store cells at 4°C in PBS overnight.

C. Immunostaining

NOTE: Count cells using a hemocytometer or alternative method.

  1. Aliquot desired number of cells into tubes or wells. (Generally, 5x105 to 1x106 cells per assay).
  2. Centrifuge cells and discard supernatant.
  3. Resuspend cells in 100 µl of diluted antibody conjugates, prepared in Antibody Dilution Buffer at a recommended dilution or as determined via titration.
  4. Incubate for 1 hr at room temperature (20-25°C). Protect from light.
  5. Wash by centrifugation in Antibody Dilution Buffer or 1X PBS. Discard supernatant. Repeat.
  6. Resuspend cells in 1X PBS and analyze on flow cytometer.

posted January 2017

revised August 2019

Protocol Id: 1344

Specificity / Sensitivity

OX40 (D1S6L) Rabbit mAb (Alexa Fluor® 488 Conjugate) recognizes endogenous levels of total OX40 protein. This antibody binds the intracellular domain of human OX40 protein.

Species Reactivity:

Human

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues near the carboxy terminus of human OX40 protein.

Background

OX40 (TNFRSF4, CD134) is a member of the tumor necrosis factor (TNF) receptor superfamily that regulates T cell activity and immune responses. The OX40 protein contains four cysteine rich domains, a transmembrane domain, and a cytoplasmic tail containing a QEE motif (1,2). OX40 is primarily expressed on activated CD4+ and CD8+ T-cells, while the OX40 ligand (OX40L, TNFSF4, CD252) is predominantly expressed on activated antigen presenting cells (3-7). The engagement of OX40 with OX40L leads to the recruitment of TNF receptor-associated factors (TRAFs) and results in the formation of a TCR-independent signaling complex. One component of this complex, PKCθ, activates the NF-κB pathway (2,8). OX40 signaling through Akt can also enhance TCR signaling directly (9). Research studies indicate that the OX40L-OX40 pathway is associated with inflammation and autoimmune diseases (10). Additional research studies show that OX40 agonists augment anti-tumor immunity in several cancer types (11,12).

  1. Croft, M. (2010) Annu Rev Immunol 28, 57-78.
  2. So, T. and Croft, M. (2012) Front Immunol 3, 133.
  3. Paterson, D.J. et al. (1987) Mol Immunol 24, 1281-90.
  4. Mallett, S. et al. (1990) EMBO J 9, 1063-8.
  5. Dürkop, H. et al. (1995) Br J Haematol 91, 927-31.
  6. Godfrey, W.R. et al. (1994) J Exp Med 180, 757-62.
  7. Al-Shamkhani, A. et al. (1997) J Biol Chem 272, 5275-82.
  8. So, T. et al. (2011) Proc Natl Acad Sci U S A 108, 2903-8.
  9. So, T. and Croft, M. (2013) Front Immunol 4, 139.
  10. Gough, M.J. and Weinberg, A.D. (2009) Adv Exp Med Biol 647, 94-107.
  11. Weinberg, A.D. et al. (2011) Immunol Rev 244, 218-31.
  12. Linch, S.N. et al. (2015) Front Oncol 5, 34.

Pathways & Proteins

Explore pathways + proteins related to this product.

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

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
Alexa Fluor is a registered trademark of Life Technologies Corporation.
The Alexa Fluor dye conjugates in this product are sold under license from Life Technologies Corporation, for research use only excluding use in combination with DNA microarrays and high content screening (HCS).
This product is provided under an intellectual property license from Life Technologies Corporation. The transfer of this product is conditioned on the buyer using the purchased product solely in research conducted by the buyer, excluding contract research or any fee for service research, and the buyer must not (1) use this product or its components for (a) diagnostic, therapeutic or prophylactic purposes; (b) testing, analysis or screening services, or information in return for compensation on a per-test basis; or (c) manufacturing or quality assurance or quality control, and/or (2) sell or transfer this product or its components for resale, whether or not resold for use in research. For information on purchasing a license to this product for purposes other than as described above, contact Life Technologies Corporation, 5791 Van Allen Way, Carlsbad, CA 92008 USA or outlicensing@lifetech.com.