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15136S 100 µl (50 tests) $199.00.0
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REACTIVITY SENSITIVITY MW (kDa) Isotype
H Endogenous Rabbit IgG
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Immunofluorescence (Immunocytochemistry)

Confocal immunofluorescent analysis of HuT 102 (left) and Jurkat (right) cells using OX40 (D1S6L) Rabbit mAb (Alexa Fluor® 647 Conjugate) (red). Blue pseudocolor = Propidium Iodide (PI)/RNase Staining Solution #4087.

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Flow Cytometry

Flow cytometric analysis of human peripheral blood mononuclear cells, untreated (left) or PHA-treated (1 μg/ml, 72 hr, 37ºC; right), using OX40 (D1S6L) Rabbit mAb (Alexa Fluor® 647 Conjugate) and co-stained with an anti-human CD4 antibody.

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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
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Flow Cytometry General Protocol

If using whole blood, please follow the Flow Cytometry Whole Blood Protocol.

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 1 L 1X PBS: add 50 ml 20X PBS to 950 ml dH2O, mix.
  2. 16% Formaldehyde (methanol free).
  3. 100% methanol.
  4. Incubation Buffer: Dissolve 0.5 g Bovine Serum Albumin (BSA) (#9998) in 100 ml 1X PBS. Store at 4°C.

B. Fixation

  1. Collect cells by centrifugation and aspirate supernatant.
  2. Resuspend cells in 0.5–1 ml 1X PBS. Add formaldehyde to obtain a final concentration of 4%.
  3. Fix for 10 min at 37°C.
  4. Chill tubes on ice for 1 min.
  5. For extracellular staining with antibodies that do not require permeabilization, proceed to immunostaining (Section D) or store cells in PBS with 0.1% sodium azide at 4°C; for intracellular staining, proceed to permeabilization (Section C).

C. Permeabilization

NOTE: This step is critical for many CST antibodies.

  1. Permeabilize cells by adding ice-cold 100% methanol slowly to pre-chilled cells, while gently vortexing, to a final concentration of 90% methanol. Alternatively, remove fix prior to permeabilization by centrifugation and resuspend in 90% methanol as described above.
  2. Incubate 30 min on ice.
  3. Proceed with immunostaining (Section D) or store cells at -20°C in 90% methanol.

D. Immunostaining

NOTE: Account for isotype matched controls for monoclonal antibodies or species matched IgG for polyclonal antibodies. Count cells using a hemocytometer or alternative method.

  1. Aliquot 0.5–1 x 106 cells into each assay tube (by volume).
  2. Add 2–3 ml incubation buffer to each tube and wash by centrifugation. Repeat.
  3. Resuspend cells in 100 µl of diluted primary antibody (prepared in incubation buffer at the recommended dilution).
  4. Incubate for 1 hr at room temperature.
  5. Wash by centrifugation in 2–3 ml incubation buffer.
  6. Resuspend cells in 0.5 ml PBS and analyze on flow cytometer; alternatively, for DNA staining, proceed to optional DNA stain (Section E).

E. Optional DNA Dye

  1. Resuspend cells in 0.5 ml of DNA dye (e.g. Propidium Iodide (PI)/RNase Staining Solution #4087).
  2. Incubate for at least 30 min at room temperature.
  3. Analyze cells in DNA staining solution on flow cytometer.

posted July 2009

revised September 2013

Flow Cytometry Whole Blood Protocol

If using cell lines, please follow the Flow Cytometry General Protocol.

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 1 L 1X PBS: add 50 ml 20X PBS to 950 ml dH2O, mix.
  2. 16% Formaldehyde (methanol free).
  3. Triton™ X-100: To prepare 50 ml of 0.1% Triton™ X-100 add 50 μl Triton™ X-100 to 50 ml 1 X PBS and mix well.
  4. 50% methanol.
  5. Incubation Buffer: Dissolve 0.5 g Bovine Serum Albumin (BSA) (#9998) in 100 ml 1X PBS. Store at 4°C.

B. Preparation of Whole Blood (fixation, lysis, and permeabilization) for Immunostaining

  1. Aliquot 100 μl fresh whole blood per assay tube.
  2. OPTIONAL: Place tubes in rack in 37°C water bath for short-term treatments with ligands, inhibitors, drugs, etc.
  3. Add 65 μl of 10% formaldehyde to each tube.
  4. Vortex briefly and let stand for 15 min at room temperature.
  5. Add 1 ml of 0.1% Triton™ X-100 to each tube.
  6. Vortex and let stand for 30 min at room temperature.
  7. Add 1 ml incubation buffer.
  8. Pellet cells by centrifugation and aspirate supernatant.
  9. Repeat steps 7 and 8.
  10. Resuspend cells in ice-cold 50% methanol in PBS (store methanol solution at -20°C until use).
  11. Incubate at least 10 min on ice.
  12. Proceed with staining or store cells at -20°C in 50% methanol.

C. Staining Using Conjugated Primary Antibodies

NOTE: Account for isotype-matched controls for monoclonal antibodies or species matched IgG for polyclonal antibodies.

  1. Add 2–3 ml incubation buffer to each tube and rinse by centrifugation. Repeat.
  2. Add primary antibodies diluted as recommended on datasheet or product webpage in incubation buffer.
  3. Incubate for 1 hr at room temperature.
  4. Wash by centrifugation in 2–3 ml incubation buffer.
  5. Resuspend cells in 0.5 ml PBS and analyze on flow cytometer.

Reference: Chow S, Hedley D, Grom P, Magari R, Jacobberger JW, Shankey TV (2005) Whole blood fixation and permeabilization protocol with red blood cell lysis for flow cytometry of intracellular phosphorylated epitopes in leukocyte subpopulations. Cytometry A 67(1), 4–17.

posted November 2008

revised September 2013

Protocol Id: 407

Product Usage Information

Application Dilutions
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.

Specificity / Sensitivity

OX40 (D1S6L) Rabbit mAb (Alexa Fluor® 647 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.

Product Description

This Cell Signaling Technology antibody is conjugated to Alexa Fluor® 647 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.


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.


Entrez-Gene Id 7293
Swiss-Prot Acc. P43489


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 contingent on the buyer using the purchased product solely in research, including use with HCS or other automated imaging applications but excluding use in combination with DNA microarrays. The buyer must not sell or otherwise transfer 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; (c) manufacturing or quality assurance or quality control, or (d) 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.

15136
OX40 (D1S6L) Rabbit mAb (Alexa Fluor® 647 Conjugate)