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15119S 100 µl (200 tests) $239.00


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H Endogenous 30 Rabbit IgG

Flow Cytometry

Flow cytometric analysis of human peripheral blood mononuclear cells, untreated (left) or PHA-treated (1 ug/ml, 72 hr; right), using CTLA-4 (D4E9I) Rabbit mAb and co-stained with a CD3 antibody. Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 647 Conjugate) #4414 was used as a secondary antibody. Analysis was performed on cells in the lymphocyte gate.

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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.

C. Staining Using Unlabeled Primary and Conjugated Secondary Antibodies

NOTE: Allow species matched IgG for polyclonal antibodies. Count cells using a hemacytometer or alternative method.

  1. Aliquot 0.5-1x106 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 minutes at room temperature.
  5. Add the primary antibody at the appropriate dilution to the assay tubes (see individual antibody data sheet for the appropriate dilution).
  6. Incubate for 30-60 minutes at room temperature.
  7. Rinse as before in Incubation Buffer by centrifugation.
  8. Resuspend cells in fluorochrome-conjugated secondary antibody*, diluted in Incubation Buffer according to the manufacturer’s recommendations.
  9. Incubate for 30 minutes at room temperature.
  10. Rinse as before in Incubation Buffer by centrifugation.
  11. Resuspend cells in 0.5 ml PBS and analyze on flow cytometer.

*Recommended Secondary Antibodies:


posted January 2009

Protocol Id: 133

Product Usage Information

Application Dilutions
Flow Cytometry 1:200

Storage: Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody.

Specificity / Sensitivity

CTLA-4 (D4E9I) Rabbit mAb 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.

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.

CTLA-4 (D4E9I) Rabbit mAb