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

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

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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).

To Purchase # 53957S
Product # Size Price
53957S
100 µl  (50 tests) $ 305

Supporting Data

REACTIVITY M
SENSITIVITY Endogenous
MW (kDa)
Isotype Rabbit IgG

Product Description

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.

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.

Protocol

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Flow Cytometry, Methanol Permeabilization Protocol for Directly Conjugated Antibodies

A. Solutions and Reagents

All reagents required for this protocol may be efficiently purchased together in our Intracellular Flow Cytometry Kit (Methanol) #13593, or individually using the catalog numbers listed below.

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.

  1. 1X Phosphate Buffered Saline (PBS): To prepare 1 L 1X PBS: add 100 ml 10X PBS (#12528) to 900 ml water mix.
  2. 4% Formaldehyde, Methanol-Free (#47746)
  3. 100% Methanol (#13604): Chill before use
  4. Antibody Dilution Buffer: Purchase ready-to-use Flow Cytometry Antibody Dilution Buffer (#13616), or prepare a 0.5% BSA PBS buffer by dissolving 0.5 g Bovine Serum Albumin (BSA) (#9998) in 100 ml 1X PBS. Store at 4°C.

NOTE: When including fluorescent cellular dyes in your experiment (including viability dyes, DNA dyes, etc.), please refer to the dye product page for the recommended protocol. Visit www.cellsignal.com/flowdyes for a listing of cellular dyes validated for use in flow cytometry.

B. Fixation

NOTE: Adherent cells or tissue should be dissociated and in single-cell suspension prior to fixation.

NOTE: Optimal centrifugation conditions will vary depending upon cell type and reagent volume. Generally, 150-300g for 1-5 minutes will be sufficient to pellet the cells.

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

NOTE: Antibodies targeting CD markers or other extracellular proteins may be added prior to fixation if the epitope is disrupted by formaldehyde and/or methanol. The antibodies will remain bound to the target of interest during the fixation and permeabilization process. However, note that some fluorophores (including PE and APC) are damaged by methanol and thus should not be added prior to permeabilization. Conduct a small-scale experiment if you are unsure.

  1. Pellet cells by centrifugation and remove supernatant.
  2. Resuspend cells in approximately 100 µl 4% formaldehyde per 1 million cells. Mix well to dissociate pellet and prevent cross-linking of individual cells.
  3. Fix for 15 min at room temperature (20-25°C).
  4. Wash by centrifugation with excess 1X PBS. Discard supernatant in appropriate waste container. Resuspend cells in 0.5-1 ml 1X PBS. Proceed to Permeabilization step.
    1. Alternatively, cells may be stored overnight at 4°C in 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. Permeabilize for a minimum of 10 min on ice.
  3. Proceed with immunostaining (Section D) or store cells at -20°C in 90% methanol.

D. Immunostaining

NOTE: Count cells using a hemocytometer or alternative method.

  1. Aliquot desired number of cells into tubes or wells. (Generally, 5x105 to 1x106 cells per assay.)
  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 primary antibody, prepared in Antibody Dilution Buffer at a recommended dilution or as determined via titration.
  4. Incubate for 1 hr at room temperature. Protect from light.
  5. Wash by centrifugation in Antibody Dilution Buffer or 1X PBS. Discard supernatant. Repeat.
  6. Resuspend cells in 200-500 µl of 1X PBS and analyze on flow cytometer.

posted July 2009

revised August 2019

Protocol Id: 407

Specificity / Sensitivity

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

Species Reactivity:

Mouse

Source / Purification

Monoclonal antibody is produced by immunizing animals with recombinant protein specific to the carboxy terminus of mouse IL-1α protein.

Background

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.

Pathways & Proteins

Explore pathways + proteins related to this product.

For Research Use Only. Not For Use In Diagnostic Procedures.

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