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15119
CTLA-4 (D4E9I) Rabbit mAb

CTLA-4 (D4E9I) Rabbit mAb #15119

APPLICATIONS

REACTIVITY SENSITIVITY MW (kDa) Isotype
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, 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

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.

CTLA-4 (D4E9I) 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.

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.

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To Purchase # 15119S

Product Number Size Price
15119S 100 µl (200 tests) $246.00.0
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