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|15149S||100 µl (50 tests)||$199.00.0|
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OX40 (D1S6L) Rabbit mAb (PE Conjugate) #15149
Gallery: OX40 (D1S6L) Rabbit mAb (PE Conjugate) #15149
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 (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).
For Research Use Only. Not For Use In Diagnostic Procedures. Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.