Upstream / Downstream

pathwayImage

Explore pathways related to this product.

Dec 31st Santa Cruz will discontinue a
large number of polyclonal products as
a result of the USDA settlement that was
made public May 19th.

Find CST Equivalent

To Purchase # 39901S

39901S 100 µl (50 tests) $299.00.0
$0.00

Questions?

Find answers on our FAQs page.

ANSWERS  

PhosphoSitePlus® Resource

  • Additional protein information
  • Analytical tools

LEARN MORE

REACTIVITY SENSITIVITY MW (kDa) Isotype
M Endogenous Rabbit IgG
Image
Image
Image
Image

Flow Cytometry

Flow cytometric analysis of EL4 cells, untreated (left) or treated with TPA #4174 (50 ng/ml), Ionomycin, Calcium Salt #9995 (500 ng/ml), and Brefeldin A #9972 (500 ng/ml, overnight; right), using IL-17A (D1X7L) Rabbit mAb (Mouse Specific) (PE Conjugate).

Learn more about how we get our images
Image
Image
Page

Flow Cytometry General Protocol

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. 16% Formaldehyde (methanol free).
  3. 0.1% Saponin in 1X PBS.
  4. Incubation Buffer: Dissolve 0.5 g Bovine Serum Albumin (BSA) (#9998) in 100 ml 1X PBS. Store at 4°C.
  5. Recommended Anti-Rabbit secondary antibodies:

B. Fixation

  1. Collect cells by centrifugation and aspirate supernatant.
  2. Resuspend cells in 0.5–1 ml 1X PBS. Add appropriate volume of 16% Formaldehyde to obtain a final concentration of 4% (e.g. 750 μl PBS + 250 μl 16% Formaldehyde).
  3. Fix for 10 min in a 37°C water bath.
  4. Chill samples on ice for 1 min.
  5. For extracellular staining with antibodies that do not require permeabilization, proceed to immunostaining (Section D) or store cells in 1X PBS with 0.1% sodium azide at 4°C; for intracellular staining, proceed to permeabilization (Section C).

C. Permeabilization

  1. Wash cells by centrifugation and resuspend in 1 ml 0.1% Saponin/1X PBS.
  2. Incubate 30 min at room temperature.
  3. Add 2 ml of 0.1% Saponin/1X PBS and wash by centrifugation.

D. Immunostaining

  1. Resuspend cells in 100 µl of primary antibody diluted in 0.1% Saponin/1X PBS. See individual antibody datasheet or product webpage for the appropriate antibody dilution.
  2. Incubate for 1 hr at room temperature.
  3. Wash by centrifugation in 2 ml 0.1% Saponin/1X PBS.
  4. Resuspend cells in fluorochrome-conjugated secondary antibody or fluorochrome-conjugated avidin, diluted in 0.1% Saponin/1X PBS at the recommended dilution.
  5. Incubate for 30 min at room temperature.
  6. Wash by centrifugation in 2 ml 0.1% Saponin/1X PBS.
  7. Resuspend cells in 0.5 ml 1X PBS and analyze on flow cytometer; alternatively, for DNA staining, proceed to optional DNA dye (Section E).

E. 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 30 min at room temperature.
  3. Analyze cells in DNA staining solution on flow cytometer.

posted June 2014

Protocol Id: 604

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

IL-17A (D1X7L) Rabbit mAb (Mouse Specific) (PE Conjugate) recognizes endogenous levels of total mouse IL-17A protein.


Species Reactivity: Mouse
Species predicted to react based on 100% sequence homology: Rat

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Val49 of mouse IL-17A protein.

Product Description

This Cell Signaling Technology antibody is conjugated to phycoerythrin (PE) and tested in-house for direct flow cytometry analysis in mouse cells. This antibody is expected to exhibit the same species cross-reactivity as the unconjugated IL-17A (D1X7L) Rabbit mAb (Mouse Specific) #13838.


The IL-17 family of cytokines consists of IL-17A-F, and their receptors include IL-17RA-RE (1). IL-17 cytokines are produced by a variety of cell types including the Th17 subset of CD4+ T cells, as well as subsets of γδ T cells, NK cells, and NKT cells (2). IL-17A and IL-17F, the most well-studied of the IL-17 cytokines, contribute to fungal and bacterial immunity by inducing expression of proinflammatory cytokines, chemokines, and antimicrobial peptides (2). In addition, IL-17A contributes to the pathogenesis of several autoimmune diseases (3). IL-17E promotes Th2 cell responses (4). The roles of IL-17B, IL-17C, and IL-17D are less clear, however these family members also appear to have the capacity to induce proinflammatory cytokines (1,5,6). IL-17 receptors have an extracellular domain, a transmembrane domain, and a SEFIR domain. They are believed to signal as homodimers, heterodimers, or multimers through their SEFIR domain by recruiting the SEFIR domain-containing adaptor Act1 (7). Unlike most cytokines that signal through Jak/STAT pathways, IL-17 signaling results in NF-κB activation (8).


1.  Gaffen, S.L. (2009) Nat Rev Immunol 9, 556-67.

2.  Iwakura, Y. et al. (2011) Immunity 34, 149-62.

3.  Hu, Y. et al. (2011) Ann N Y Acad Sci 1217, 60-76.

4.  Fort, M.M. et al. (2001) Immunity 15, 985-95.

5.  Yamaguchi, Y. et al. (2007) J Immunol 179, 7128-36.

6.  Li, H. et al. (2000) Proc Natl Acad Sci U S A 97, 773-8.

7.  Chang, S.H. et al. (2006) J Biol Chem 281, 35603-7.

8.  Shalom-Barak, T. et al. (1998) J Biol Chem 273, 27467-73.


Entrez-Gene Id 16171
Swiss-Prot Acc. Q62386


For Research Use Only. Not For Use In Diagnostic Procedures.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.

39901
IL-17A (D1X7L) Rabbit mAb (Mouse Specific) (PE Conjugate)