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15148S 100 µl (50 tests) $199.00


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H Endogenous Rabbit IgG

Flow Cytometry

Flow cytometric analysis of human peripheral blood mononuclear cells, untreated (left) or PHA-treated (1 μg/ml, 48 hr, 37ºC; right), using OX40 (D4N3K) Rabbit mAb (Pacific Blue™ Conjugate) and co-stained with an anti-human CD4 antibody.

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Flow Cytometry

A. Solutions and Reagents

  1. 1X Phosphate Buffered Saline (PBS): Dissolve 8 g NaCl, 0.2 g KCl, 1.44 g Na2HPO4 and 0.24 g KH2PO4 in 800 mL distilled water (dH2O). Adjust the pH to 7.4 with HCl and the volume to 1 liter. Store at room temperature.
  2. Formaldehyde (methanol free).
  3. Incubation Buffer: Dissolve 0.5 g bovine serum albumin (BSA) in 100mL 1X PBS. Store at 4°C.

B. 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 10 minutes at 37°C.
  4. Add 5 ml PBS and rinse by centrifugation.
  5. Resuspend cells in 5 ml PBS.
  6. Proceed with staining or store cells at +4°C in PBS with 0.1% sodium azide.

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 rinse by centrifugation. Repeat.
  3. Resuspend cells in 100 µl incubation buffer per assay tube.
  4. Block in Incubation Buffer for 10 min at room temperature.
  5. Add fluorochrome-conjugated primary antibody at the appropriate dilution to the assay tubes (see individual antibody datasheet or product webpage for the appropriate dilution).
  6. Incubate for 1 hr at room temperature.
  7. Wash by centrifugation in 2–3 ml incubation buffer.
  8. Resuspend cells in 0.5 ml PBS and analyze on flow cytometer; alternatively, for DNA staining, proceed to optional DNA stain (Section E).

posted January 2009

Protocol Id: 180

Product Usage Information

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 antibody. Protect from light. Do not freeze.

Specificity / Sensitivity

OX40 (D4N3K) Rabbit mAb (Pacific Blue™ Conjugate) recognizes endogenous levels of total OX40 protein. This antibody binds the extracellular domain of human OX40 protein.

Species Reactivity: Human

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Gln156 of human OX40 protein.

Product Description

This Cell Signaling Technology antibody is conjugated to Pacific Blue™ fluorescent dye and tested in-house for direct flow cytometry in human cells. The antibody is expected to exhibit the same species cross-reactivity as the unconjugated antibody OX40 (D4N3K) Rabbit mAb  #15122.

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.
Pacific Blue is a trademark of Molecular Probes, Inc.

OX40 (D4N3K) Rabbit mAb (Pacific Blue™ Conjugate)