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15666
CD28 (CD28.2) Mouse mAb (FITC Conjugate)
Antibody Conjugates

CD28 (CD28.2) Mouse mAb (FITC Conjugate) #15666

APPLICATIONS

REACTIVITY SENSITIVITY MW (kDa) Isotype
H Endogenous Mouse IgG1, kappa
Flow Cytometry

Flow cytometric analysis of live human peripheral blood mononuclear cells using CD28 (CD28.2) Mouse mAb (FITC Conjugate) (solid line) compared to concentration-matched Mouse (MOPC-21) mAb IgG1 Isotype Control (FITC Conjugate) #97146 (dashed line).

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Flow Cytometry

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. Incubation Buffer: Dissolve 0.5 g Bovine Serum Albumin (BSA) (#9998) in 100 ml 1X PBS. Store at 4°C.

B. Immunostaining

  1. Count cells using a hemacytometer or alternative method.
  2. Aliquot 0.5-1x106 cells into each assay tube (by volume).
  3. Add 2-3 ml Incubation Buffer to each tube and rinse by centrifugation. Repeat.
  4. Resuspend cells in 100 µl of diluted primary antibody (prepared in incubation buffer at the recommended dilution).
  5. Incubate for 1 hr at room temperature.
  6. Wash by centrifugation in 2-3 ml incubation buffer.
  7. Resuspend cells in the appropriate volume of PBS and analyze on flow cytometer.

posted June 2017

Protocol Id: 1504

Application Dilutions
Flow Cytometry 1:20
Storage:

Supplied in 10 mM NaH2PO4, 150 mM NaCl, 0.09% NaN3, 0.1% gelatin, pH7.2. This product is stable for 6 months when stored at 4ºC. Do not aliquot the antibody. Protect from light. Do not freeze.

CD28 (CD28.2) Mouse mAb (FITC Conjugate) recognizes endogenous levels of total CD28 protein. This antibody detects an epitope within the extracellular domain.

Species Reactivity:

Human

This monoclonal antibody was purified from tissue culture supernatant via affinity chromatography. The purified antibody was conjugated under optimal conditions, with unreacted dye removed from the preparation.

This Cell Signaling Technology antibody is conjugated to FITC and tested in-house for direct flow cytometric analysis in human cells.

CD28 is a transmembrane glycoprotein expressed by T cells as well as some other hematopoietic cells (1, 2). T cell activation requires T cell receptor (TCR) recognition of antigen presented in the context of MHC molecules. CD28 acts as a T cell costimulatory receptor, and interaction of CD28 with its ligands CD80 or CD86 provides the second signal required for naïve T cell activation (3-5). Activation of naïve T cells in the absence of CD28 stimulation can result in a state of T cell anergy, or unresponsiveness (3). CD28 signals through cytoplasmic phospho-tyrosine motifs that bind several SH2 or SH3 domain-containing proteins involved in T cell activation (2). Recently, CD28 was demonstrated to be a preferred target of PD-1-mediated dephosphorylation. Consistently, CD28 expression was required for T cell proliferation following PD-1 blockade and CD28 stimulation was required for effective anti-PD-1 cancer immunotherapy in mice (6, 7). Several CD28 isoforms are produced by alternative splicing (8).

  1. Aruffo, A. and Seed, B. (1987) Proc Natl Acad Sci U S A 84, 8573-7.
  2. Esensten, J.H. et al. (2016) Immunity 44, 973-88.
  3. Harding, F.A. et al. (1992) Nature 356, 607-9.
  4. Azuma, M. et al. (1993) Nature 366, 76-9.
  5. Linsley, P.S. et al. (1990) Proc Natl Acad Sci U S A 87, 5031-5.
  6. Hui, E. et al. (2017) Science 355, 1428-1433.
  7. Kamphorst, A.O. et al. (2017) Science 355, 1423-1427.
  8. Magistrelli, G. et al. (1999) Biochem Biophys Res Commun 259, 34-7.
Entrez-Gene Id
940
Swiss-Prot Acc.
P10747
For Research Use Only. Not For Use In Diagnostic Procedures.

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.

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To Purchase # 15666S

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Product # Size Price
15666S
500 µl  (100 tests) $ 129.0