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16900
Neutrophil Elastase (E9C9L) XP® Rabbit mAb (Alexa Fluor® 488 Conjugate)
Antibody Conjugates

Neutrophil Elastase (E9C9L) XP® Rabbit mAb (Alexa Fluor® 488 Conjugate) #16900

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APPLICATIONS

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

Flow cytometric analysis of Jurkat cells (blue) and U-937 cells (green) using Neutrophil Elastase (E9C9L) XP® Rabbit mAb (Alexa Fluor® 488 Conjugate) (solid lines) or concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control (Alexa Fluor® 488 Conjugate) #2975 (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.

Neutrophil Elastase (E9C9L) XP® Rabbit mAb (Alexa Fluor® 488 Conjugate) recognizes endogenous levels of total neutrophil elastase protein.

Species Reactivity:

Human

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues near the carboxy terminus of human neutrophil elastase protein.

This Cell Signaling Technology antibody is conjugated to Alexa Fluor® 488 fluorescent dye 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 Neutrophil Elastase (E9C9L) XP® Rabbit mAb #89241.

Neutrophil elastase is hematopoietic serine protease that belongs to the chymotrypsin superfamily and plays a critical role in the innate immune function of mature neutrophils and monocytes (1,2). Neutrophil elastase is actively synthesized as an inactive zymogen in myelocytic precursor cells of the bone marrow, which then undergoes activation by limited proteolysis and sorting to primary (azurophil) storage granules of mature neutrophil granulocytes for regulated release (3,4). Research studies have shown that neutrophils play a significant role in mediating the inflammatory response through the release of neutrophil elastase, which activates pro-inflammatory cytokines and degrades components of the extracellular matrix and Gram-negative bacteria (5). Mutations in the gene encoding neutrophil elastase, ELA2, have been implicated in hematological diseases such as cyclic and severe congenital neutropenia, which is characterized by defects in promyelocyte maturation (6,7).

  1. Belaaouaj, A. et al. (2000) Science 289, 1185-8.
  2. Tkalcevic, J. et al. (2000) Immunity 12, 201-10.
  3. Gullberg, U. et al. (1999) J Immunol Methods 232, 201-10.
  4. Takahashi, H. et al. (1988) J Biol Chem 263, 2543-7.
  5. Adkison, A.M. et al. (2002) J Clin Invest 109, 363-71.
  6. Horwitz, M. et al. (1999) Nat Genet 23, 433-6.
  7. Bellanné-Chantelot, C. et al. (2004) Blood 103, 4119-25.
Entrez-Gene Id
1991
Swiss-Prot Acc.
P08246
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.
This product is provided under an intellectual property license from Life Technologies Corporation. The transfer of this product is conditioned on the buyer using the purchased product solely in research conducted by the buyer, excluding contract research or any fee for service research, and the buyer must not (1) use this product or its components for (a) diagnostic, therapeutic or prophylactic purposes; (b) testing, analysis or screening services, or information in return for compensation on a per-test basis; or (c) manufacturing or quality assurance or quality control, and/or (2) sell or transfer this product or its components for resale, whether or not resold for use in research. For information on purchasing a license to this product for purposes other than as described above, contact Life Technologies Corporation, 5791 Van Allen Way, Carlsbad, CA 92008 USA or outlicensing@lifetech.com.

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製品番号 サイズ 価格 在庫
16900S
100 µl  (50 tests)

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