Upstream / Downstream
Explore pathways related to this product.
the purchase of 3 or more products
To Purchase # 15132S
|15132S||100 µl (50 tests)||$199.00.0|
Find answers on our FAQs page.
- Additional protein information
- Analytical tools
CTLA-4 (D4E9I) Rabbit mAb (PE Conjugate) #15132
Flow cytometric analysis of human peripheral blood mononuclear cells, untreated (left) or PHA-treated (1 μg/ml, 72 hr; right), using CTLA-4 (D4E9I) Rabbit mAb (PE Conjugate) and co-stained with a CD3 antibody. Analysis was performed on cells in the lymphocyte gate.Learn more about how we get our images
Gallery: CTLA-4 (D4E9I) Rabbit mAb (PE Conjugate) #15132
Flow Cytometry, Extracellular Epitope Protocol for Direct Conjugates
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.
- Formaldehyde (methanol free).
- Incubation Buffer: Dissolve 0.5 g bovine serum albumin (BSA) (#9998) in 100mL 1X PBS. Store at 4°C.
NOTE: If live cell staining is desired, proceed to Section C.
- Collect cells by centrifugation and aspirate supernatant.
- Resuspend cells in 0.5-1 ml PBS. Add formaldehyde to obtain a final concentration of 4% formaldehyde.
- Fix for 15 minutes at room temperature.
- Wash by centrifugation with excess 1X PBS. Discard supernatant in appropriate waste container. Resuspend cells in 1X PBS.
- Proceed with staining or store cells at +4°C in PBS with 0.1% sodium azide.
NOTE: Count cells using a hemocytometer or alternative method.
- Aliquot desired number of cells into tubes or wells.
- If necessary, centrifuge to remove excess PBS.
- Resuspend cells in 100 µl of diluted antibody conjugate (prepared in incubation buffer at the recommended dilution).
- Incubate for 30-60 minutes at room temperature.
- Wash by centrifugation in incubation buffer. Discard supernatant. Repeat.
- Resuspend cells in 1X PBS and analyze on flow cytometer; alternatively, for DNA staining, proceed to Section D.
D. Optional DNA Dye
- Resuspend cells in 0.5 ml of DNA dye (e.g. Propidium Iodide (PI)/RNase Staining Solution #4087).
- Incubate for at least 5 min at room temperature.
- Analyze cells in DNA staining solution on flow cytometer.
posted January 2009
revised June 2017
CTLA-4 (D4E9I) (PE Conjugate) Rabbit mAb recognizes endogenous levels of total CTLA-4 protein.Species Reactivity: Human
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Asp100 of human CTLA-4 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 CTLA-4 (D4E9I) Rabbit mAb #15119.
Cytotoxic T-lymphocyte protein 4 (CTLA-4, CD152) is an Ig superfamily member that negatively regulates early T cell activation (1-4). The CTLA-4 protein is primarily expressed on T cells, including CD8+ cytotoxic T cells, CD4+ helper T cells, and CD4+/FoxP3+ regulatory T cells (1,2). CTLA-4 protein competes with CD28 for B7.1 (CD80) and B7.2 (CD86) binding at the cell surface, which results in the down regulation of T cell activity (5). The activation of SHP-2 and PP2A downstream of CTLA-4 attenuates TCR signaling (6). Research studies indicate that CTLA4 knockout mice display lymphoproliferative disorders leading to early death, confirming the role of CTLA-4 as a negative regulator of T cells (7). Mutations in the corresponding CTLA4 gene are associated with multiple disorders, including insulin-dependent diabetes mellitus, Graves disease, Hashimoto thyroiditis, celiac disease, systemic lupus erythematosus, and type V autoimmune lymphoproliferative syndrome (8,9). Additional studies demonstrate that CTLA-4 blockade is an effective strategy for tumor immunotherapy (10-12).
For Research Use Only. Not For Use In Diagnostic Procedures. Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.