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15149
OX40 (D1S6L) Rabbit mAb (PE Conjugate)

OX40 (D1S6L) Rabbit mAb (PE Conjugate) #15149

APPLICATIONS

REACTIVITY SENSITIVITY MW (kDa) Isotype
H Endogenous Rabbit IgG
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 (PE Conjugate) and co-stained with an anti-human CD4 antibody.

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Flow Cytometry Triton™ X-100 Permeabilization Protocol - Conjugated Antibody

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. Adjust pH to 8.0.
  2. Formaldehyde (methanol free).
  3. Incubation Buffer (1X PBS / 0.5% BSA): Dissolve 0.5 g bovine serum albumin (BSA) in 100 mL 1X PBS. Store at 4°C.
  4. Permeabilization Buffer (1X PBS / 0.3% Triton™ X-100 / 0.5% BSA): To prepare 10 ml, add 30 µl Triton™ X-100 to 10 ml Incubation Buffer.

B. Fixation and Permabilization

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

  1. Collect cells by centrifugation and aspirate supernatant.
  2. Resuspend cells briefly in 0.5-1 ml PBS. Add formaldehyde to a final concentration of 2-4% formaldehyde.
  3. Fix for 15 minutes at room temperature (20-25 °C).
  4. Wash by centrifugation with excess 1X PBS. Discard supernatant in appropriate waste container.
  5. Resuspend cells in 1 ml Permeabilization Buffer.
  6. Incubate for 10 minutes at room temperature.
  7. Proceed with staining or store cells at +4°C in PBS with 0.1% sodium azide.

C. Staining Using Conjugated Primary Antibody

NOTE: Count cells using a hemocytometer or alternative method.

  1. Aliquot desired number of cells into tubes or wells (suggested range of 0.5x106 to 1x106 cells).
  2. Centrifuge cells and discard supernatant.
  3. Resuspend cells in 100 µl of diluted conjugated primary antibody (prepared in Incubation Buffer at the recommended dilution).
  4. Incubate for 30-60 min at room temperature (20-25 °C). Protect from light.
  5. Wash by centrifugation in 1X PBS. Discard supernatant. Repeat.
  6. Resuspend cells in 1X PBS and analyze on flow cytometer; alternatively, for DNA staining, proceed to optional DNA stain (Section D).

D. Optional DNA Dye

  1. Resuspend cells in 0.1-0.5 ml of DNA dye (e.g. Propidium Iodide (PI)/RNase Staining Solution #4087).
  2. Incubate for at least 5 min at room temperature.
  3. Analyze cells in DNA staining solution on flow cytometer.

posted January 2017

revised May 2018

Protocol Id: 1344

Application Dilutions
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 antibodies. Protect from light. Do not freeze.

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

Species Reactivity:

Human

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

This Cell Signaling Technology antibody is conjugated to phycoerythrin (PE) and tested in-house for direct flow cytometry analysis in human cells. The 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.

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Product # Size Price
15149S
100 µl (50 tests) $ 206.0

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