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15162S 100 µl (50 tests) $199.00.0
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REACTIVITY SENSITIVITY MW (kDa) Isotype
H Endogenous Rabbit IgG
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Flow Cytometry

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 (Alexa Fluor® 488 Conjugate) and co-stained with a CD3 antibody. Analysis was performed on cells in the lymphocyte gate.

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Flow Cytometry, Extracellular Epitope Protocol for Rabbit Antibodies

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.
  2. Formaldehyde (methanol free).
  3. Incubation Buffer: Dissolve 0.5 g bovine serum albumin (BSA) (#9998) in 100 ml 1X PBS. Store at 4°C.
  4. Recommended Anti-Rabbit secondary antibodies:

B. Fixation

NOTE: If live cell staining is desired, proceed to Section C.

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

C. Immunostaining

NOTE: Count cells using a hemocytometer or alternative method.

  1. Aliquot desired number of cells into tubes or wells.
  2. If necessary, centrifuge to remove excess PBS.
  3. Resuspend cells in 100 µl of diluted primary antibody (prepared in incubation buffer at the recommended dilution).
  4. Incubate for 30-60 minutes at room temperature.
  5. Wash by centrifugation in incubation buffer. Discard supernatant. Repeat.
  6. Resuspend cells in fluorochrome-conjugated secondary antibody at the recommended dilution.
  7. Incubate for 30 minutes at room temperature.
  8. Wash by centrifugation in incubation buffer. Discard supernatant. Repeat.
  9. Resuspend cells in 1X PBS and analyze on flow cytometer; alternatively, for DNA staining, proceed to Section D.

D. Optional DNA Dye

  1. Resuspend cells in 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 2009

revised June 2017

Protocol Id: 133

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

CTLA-4 (D4E9I) Rabbit mAb (Alexa Fluor® 488 Conjugate) recognizes endogenous levels of total CTLA-4 protein.


Species Reactivity: Human

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Asp100 of human CTLA-4 protein.

Product Description

This Cell Signaling Technology antibody is conjugated to Alexa Fluor® 488 fluorescent dye and tested in-house for direct flow cytometric analysis in human cells. This 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).


1.  Brunet, J.F. et al. (1987) Nature 328, 267-70.

2.  Brunet, J.F. et al. (1988) Immunol Rev 103, 21-36.

3.  Dariavach, P. et al. (1988) Eur J Immunol 18, 1901-5.

4.  Linsley, P.S. (1995) J Exp Med 182, 289-92.

5.  Collins, A.V. et al. (2002) Immunity 17, 201-10.

6.  Rudd, C.E. et al. (2009) Immunol Rev 229, 12-26.

7.  Waterhouse, P. et al. (1995) Science 270, 985-8.

8.  Romo-Tena, J. et al. (2013) Autoimmun Rev 12, 1171-6.

9.  Wang, J. et al. (2014) PLoS One 9, e85982.

10.  Egen, J.G. et al. (2002) Nat Immunol 3, 611-8.

11.  Hodi, F.S. et al. (2003) Proc Natl Acad Sci U S A 100, 4712-7.

12.  Pardoll, D.M. (2012) Nat Rev Cancer 12, 252-64.


Entrez-Gene Id 1493
Swiss-Prot Acc. P16410


For Research Use Only. Not For Use In Diagnostic Procedures.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
Alexa Fluor is a registered trademark of Life Technologies Corporation.
This product is provided under an intellectual property license from Life Technologies Corporation. The transfer of this product is contingent on the buyer using the purchased product solely in research, including use with HCS or other automated imaging applications but excluding use in combination with DNA microarrays. The buyer must not sell or otherwise transfer this product or its components for (a) diagnostic, therapeutic or prophylactic purposes; (b) testing, analysis or screening services, or information in return for compensation on a per-test basis; (c) manufacturing or quality assurance or quality control, or (d) resale, whether or not resold for use in research. For information on purchasing a license to this product for purposes other than as described above, contact Life Technologies Corporation, 5791 Van Allen Way, Carlsbad, CA 92008 USA or outlicensing@lifetech.com.

15162
CTLA-4 (D4E9I) Rabbit mAb (Alexa Fluor® 488 Conjugate)