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13627
IL-1β (D3U3E) Rabbit mAb (PE Conjugate)

IL-1β (D3U3E) Rabbit mAb (PE Conjugate) #13627

APPLICATIONS

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

Flow cytometric analysis of THP-1 cells, untreated (blue) or LPS-treated (100 ng/ml, 3 hr; green), using IL-1β (D3U3E) Rabbit mAb (PE Conjugate).

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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 antibodies. Protect from light. Do not freeze.

IL-1β (D3U3E) Rabbit mAb (PE Conjugate) recognizes endogenous levels of total IL-1β protein and can detect up to 100 pg of recombinant mature IL-1β. This antibody is not observed to detect endogenous levels of mature IL-1β.

Species Reactivity:

Human

Monoclonal antibody is produced by immunizing animals with recombinant human IL-1β protein.

This Cell Signaling Technology antibody is conjugated to phycoerythrin (PE) and tested in-house for direct flow cytometry analysis in human cells. The antibody is expected to exhibit the same species cross-reactivity as the unconjugated IL-1β (D3U3E) Rabbit mAb #12703.

Interleukin-1β (IL-1β), one of the major caspase-1 targets, is a multifunctional cytokine that is involved in a host of immune and proinflammatory responses (1). It is produced primarily by activated monocytes and macrophages. It signals through various adaptor proteins and kinases that lead to activation of numerous downstream targets (2-6). Human IL-1β is synthesized as a 31 kDa precursor. To gain activity, the precursor must be cleaved by caspase-1 between Asp116 and Ala117 to yield a 17 kDa mature form (7,8). Detection of the 17 kDa mature form of IL-1β is a good indicator of caspase-1 activity.

  1. Dinarello, C.A. (1998) Int Rev Immunol 16, 457-99.
  2. Burns, K. et al. (1998) J Biol Chem 273, 12203-9.
  3. Cao, Z. et al. (1996) Nature 383, 443-6.
  4. Cao, Z. et al. (1996) Science 271, 1128-31.
  5. Wesche, H. et al. (1997) Immunity 7, 837-47.
  6. Ninomiya-Tsuji, J. et al. (1999) Nature 398, 252-6.
  7. Thornberry, N.A. et al. (1992) Nature 356, 768-74.
  8. Cerretti, D.P. et al. (1992) Science 256, 97-100.
Entrez-Gene Id
3553
Swiss-Prot Acc.
P01584
For Research Use Only. Not For Use In Diagnostic Procedures.

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

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To Purchase # 13627S

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Product # Size Price
13627S
100 µl (50 tests) $ 299.0

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