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91979
S100A9 (D5O6O) Rabbit mAb (PE Conjugate)

S100A9 (D5O6O) Rabbit mAb (PE Conjugate) #91979

APPLICATIONS

REACTIVITY SENSITIVITY MW (kDa) Isotype
H Endogenous Rabbit IgG
Flow Cytometry

Flow cytometric analysis of Jurkat (blue) and THP-1 (green) cells using S100A9 (D5O6O) Rabbit mAb (PE Conjugate) (solid lines) or concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control (PE Conjugate) #5742 (dashed lines).

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Flow Cytometry, Methanol Permeabilization Protocol for Direct Conjugates

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

B. Fixation

NOTE: If using whole blood, lyse red blood cells and wash by centrifugation prior to fixation.

  1. Collect cells by centrifugation and aspirate supernatant.
  2. Resuspend cells in 0.5-1 ml 1X PBS. Add formaldehyde to obtain a final concentration of 4%.
  3. Fix for 15 min at room temperature.
  4. Wash by centrifugation with excess 1X PBS. Discard supernatant in appropriate waste container. Resuspend cells in 0.5-1 ml 1X PBS.

C. Permeabilization

  1. Permeabilize cells by adding ice-cold 100% methanol slowly to pre-chilled cells, while gently vortexing, to a final concentration of 90% methanol.
  2. Incubate 30 min on ice.
  3. Proceed with immunostaining (Section D) or store cells at -20°C in 90% methanol.

D. Immunostaining

  1. Aliquot desired number of cells into tubes or wells.
  2. Wash cells by centrifugation in excess 1X PBS to remove methanol. Discard supernatant in appropriate waste container. Repeat if necessary.
  3. Resuspend cells in 100 µl of diluted antibody conjugate (prepared in incubation buffer at the recommended dilution).
  4. Incubate for 1 hr at room temperature. Protect from light.
  5. Wash by centrifugation in incubation buffer. Discard supernatant. Repeat.
  6. Resuspend cells in 1X PBS and analyze on flow cytometer; alternatively, for DNA staining, proceed to optional DNA stain (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 5 min at room temperature.
  3. Analyze cells in DNA staining solution on flow cytometer.

posted July 2009

revised June 2017

Protocol Id: 407

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.

S100A9 (D5O6O) Rabbit mAb (PE Conjugate) recognizes endogenous levels of total S100A9 protein.

Species Reactivity:

Human

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Pro107 of human S100A9 protein.

This Cell Signaling Technology antibody is conjugated to phycoerythrin (PE) 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 S100A9 (D5O6O) Rabbit mAb #72590.

S100A8 and S100A9 are calcium-binding proteins that form a noncovalent heterodimer present in monocytes, neutrophils, macrophages, and some epithelial cells (1, 2). S100A8 and S100A9 are secreted by a tubulin-dependent mechanism during inflammatory conditions and have antimicrobial and chemotactic functions (3-5). Extracellular S100A8/S100A9 also induces an inflammatory response in endothelial cells, including induction of proinflammatory chemokines and adhesion molecules and increased vascular permeability (6). S100A8/S100A9 induces and recruits myeloid-derived suppressor cells (MDSC) in tumor-bearing mice (7). MDSC produce additional S100A8/S100A9 themselves, resulting in a positive feedback mechanism that sustains MDSC accumulation (7). S100A8/S100A9 is also highly expressed in psoriatic skin, where it directly upregulates transcription of complement protein C3, which contributes to disease (8). In addition, tumor-infiltrating myeloid cells induce expression of S100A8 and S100A9 in cancer cells, which increases invasiveness and metastasis (9).

  1. Odink, K. et al. Nature 330, 80-2.
  2. Edgeworth, J. et al. (1991) J Biol Chem 266, 7706-13.
  3. Rammes, A. et al. (1997) J Biol Chem 272, 9496-502.
  4. Steinbakk, M. et al. (1990) Lancet 336, 763-5.
  5. Ryckman, C. et al. (2003) J Immunol 170, 3233-42.
  6. Viemann, D. et al. (2005) Blood 105, 2955-62.
  7. Sinha, P. et al. (2008) J Immunol 181, 4666-75.
  8. Schonthaler, H.B. et al. (2013) Immunity 39, 1171-81.
  9. Lim, S.Y. et al. (2016) Oncogene 35, 5735-5745.
Entrez-Gene Id
6280
Swiss-Prot Acc.
P06702
For Research Use Only. Not For Use In Diagnostic Procedures.

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

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To Purchase # 91979S

Product Number Size Price
91979S 100 µl (50 tests) $299.00.0
Quantity Subtotal
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