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OX40 (D1S6L) Rabbit mAb (Pacific Blue™ Conjugate) #15147
Gallery: OX40 (D1S6L) Rabbit mAb (Pacific Blue™ Conjugate) #15147
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.
- 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.
- Formaldehyde (methanol free).
- Incubation Buffer (1X PBS / 0.5% BSA): Dissolve 0.5 g bovine serum albumin (BSA) in 100 mL 1X PBS. Store at 4°C.
- 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
- Collect cells by centrifugation and aspirate supernatant.
- Resuspend cells briefly in 0.5-1 ml PBS. Add formaldehyde to a final concentration of 2-4% formaldehyde.
- Fix for 10 minutes at 37°C.
- Add 5 ml PBS and rinse by centrifugation.
- Resuspend cells in 1 ml Permeabilization Buffer.
- Incubate for 10 minutes at room temperature.
- 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.
- Aliquot 0.5-1x106 cells into each assay tube (by volume).
- If necessary, add 2-3 ml Incubation Buffer to each tube and rinse by centrifugation.
- Add the conjugated primary antibody at the appropriate dilution to the assay tubes (see individual antibody data sheet for the appropriate dilution).
- Incubate for 30-60 minutes at room temperature.
- Rinse as before in Incubation Buffer by centrifugation.
- Resuspend cells in 0.5 ml PBS and analyze on flow cytometer.
posted January 2017
OX40 (D1S6L) Rabbit mAb (Pacific Blue™ 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 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 (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).
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.