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53957
IL-1α (D4F3S) Rabbit mAb (Mouse Specific) (PE Conjugate)

IL-1α (D4F3S) Rabbit mAb (Mouse Specific) (PE Conjugate) #53957

APPLICATIONS

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

Flow cytometric analysis of Raw 264.7 cells, untreated (blue) or treated with LPS #14011 (5 μg/mL, overnight; green), using IL-1α (D4F3S) Rabbit mAb (Mouse Specific) (PE Conjugate) (solid lines) or a concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control (PE Conjugate) #3900 (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.

IL-1α (D4F3S) Rabbit mAb (Mouse Specific) (PE Conjugate) recognizes endogenous levels of total IL-1α protein.

Species Reactivity:

Mouse

Monoclonal antibody is produced by immunizing animals with recombinant protein specific to the carboxy terminus of mouse IL-1α 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 IL-1α (D4F3S) Rabbit mAb (Mouse Specific) #50794.

Interleukin 1 alpha (IL-1a) belongs to the IL-1 family of cytokines with 11 members including IL-1b. IL-1a is expressed in many cell types of both hematopoietic and non-hematopoietic origins under steady state, and its expression can be increased in response to appropriate stimuli (1,2). Like IL-1b, IL-1a is also synthesized as a precursor (pro-IL-1a) and can be cleaved into smaller mature forms. However, both pro-IL-1a and the cleaved form of IL-1a are biologically active and can activate the signaling pathway through the membrane receptor IL-1R1. IL-1a is active both as a secreted form and as a membrane-bound form. Due to such characteristics, passive leakage of IL-1a from dying cells can activate inflammation, leading some researchers to consider IL-1a as a key “alarmin in the cell” that alerts the host to damage or injury (3,4). In addition, IL-1a can also enter the nucleus to modulate transcription (5,6).

  1. Garlanda, C. et al. (2013) Immunity 39, 1003-18.
  2. Palomo, J. et al. (2015) Cytokine 76, 25-37.
  3. Bertheloot, D. and Latz, E. (2017) Cell Mol Immunol 14, 43-64.
  4. Di Paolo, N.C. and Shayakhmetov, D.M. (2016) Nat Immunol 17, 906-13.
  5. Lamacchia, C. et al. (2013) Cytokine 63, 135-44.
  6. Rider, P. et al. (2013) Semin Immunol 25, 430-8.
Entrez-Gene Id
16175
Swiss-Prot Acc.
P01582
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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Product # Size Price
53957S
100 µl (50 tests) $ 299.0