IL-5 Antibody, anti-human

Name: IL-5 Antibody, anti-human
Availability: InStock

IL-5 Antibody, anti-humanExtended Validation

Clone:

JES1-39D10

Type of antibody:

Primary antibodies

Isotype:

rat IgG2aκ

Applications:

ICFC

Alternative names:

EDF, TRF

Intracellular flow cytometry applications forIL-5 Antibody, anti-human

APC
PE
Vio B515
Vio R667

Conjugate:

APC

Recommended dilution:

1:50

Remark:

The antibody is suited for staining of formaldehyde-fixed cells.

Tested:

QC tested

Products used:

130-127-385, 130-127-364

Protocols:

Intracellular flow cytometry staining protocol (Inside Stain Kit 1:50)

Description:

Rhesus monkey PBMCs were incubated with or without PMA/ionomycin and intracellularly stained with Anti-IL-5-PE. (Data were kindly provided by Dr. Paula Acierno, Harvard Medical School, Boston, USA.)

Extended validation for IL-5 Antibody, anti-human

Specificity

Epitope competition

In order to compare the epitope specificity of an antibody, the clone being used is compared with other known clones recognizing the same antigen in a competition assay.

Other clones	Overlap in epitope recognition with JES1-39D10
REA1025	++
TRFK5	++
Cells were incubated with an excess of purified unconjugated IL-5 (JES1-39D10) antibody followed by staining with fluorochrome-conjugated antibodies of other known clones against the same marker. Based on the fluorescence signal obtained, the clones were identified as recognizing completely overlapping (++), partially overlapping (+), or completely different epitopes (-) of the marker.

Sensitivity

Performance comparison

Selected fluorochrome conjugated antibodies from Miltenyi Biotec were compared to commercially available hybridoma clones in flow cytometry analysis.

Flow cytometric comparison of different clones for IL-5. Human peripheral blood mononuclear cells (PBMCs) were stimulated with 20 ng/mL Phorbol 12-myristate 13-acetate (PMA) and 1 µg/ml Ionomycin for 1 hour, followed by an incubation with 1µg/mL Brefeldin A for 5 hours. Cells were first stained with Viobility™ Fixable Dye followed by a suitable counterstaining. Cells were then fixed and permeabilized followed by a staining with IL-5 antibodies. As a control, IL-5 antibody staining was omitted and cells were measured in the same channels. Flow cytometry was performed with the MACSQuant^® Analyzer. CD4+ cells were pregated for the analysis. Cell debris, dead cells, and cell doublets were excluded from the analysis based on scatter signals and Viobility 405/520 Fixable Dye fluorescence. No FcR Blocking Reagent was used. The recommended titers of respective antibodies from different suppliers were used.

Flow cytometric comparison of different clones for IL-5. Human peripheral blood mononuclear cells (PBMCs) were stimulated with 20 ng/mL Phorbol 12-myristate 13-acetate (PMA) and 1 µg/ml Ionomycin for 1 hour, followed by an incubation with 1µg/mL Brefeldin A for 5 hours. Cells were first stained with Viobility™ Fixable Dye followed by a suitable counterstaining. Cells were then fixed and permeabilized followed by a staining with IL-5 antibodies. As a control, IL-5 antibody staining was omitted and cells were measured in the same channels. Flow cytometry was performed with the MACSQuant^® Analyzer. CD4+ cells were pregated for the analysis. Cell debris, dead cells, and cell doublets were excluded from the analysis based on scatter signals and Viobility 405/520 Fixable Dye fluorescence. No FcR Blocking Reagent was used. The recommended titers of respective antibodies from different suppliers were used.

Flow cytometric comparison of different clones for IL-5. Human peripheral blood mononuclear cells (PBMCs) were stimulated with 20 ng/mL Phorbol 12-myristate 13-acetate (PMA) and 1 µg/ml Ionomycin for 1 hour, followed by an incubation with 1µg/mL Brefeldin A for 5 hours. Cells were first stained with Viobility™ Fixable Dye followed by a suitable counterstaining. Cells were then fixed and permeabilized followed by a staining with IL-5 antibodies. As a control, IL-5 antibody staining was omitted and cells were measured in the same channels. Flow cytometry was performed with the MACSQuant^® Analyzer. CD4+ cells were pregated for the analysis. Cell debris, dead cells, and cell doublets were excluded from the analysis based on scatter signals and Viobility 405/520 Fixable Dye fluorescence. No FcR Blocking Reagent was used. The recommended titers of respective antibodies from different suppliers were used.

Flow cytometric comparison of different clones for IL-5. Human peripheral blood mononuclear cells (PBMCs) were stimulated with 20 ng/mL Phorbol 12-myristate 13-acetate (PMA) and 1 µg/ml Ionomycin for 1 hour, followed by an incubation with 1µg/mL Brefeldin A for 5 hours. Cells were first stained with Viobility™ Fixable Dye followed by a suitable counterstaining. Cells were then fixed and permeabilized followed by a staining with IL-5 antibodies. As a control, IL-5 antibody staining was omitted and cells were measured in the same channels. Flow cytometry was performed with the MACSQuant^® Analyzer. CD4+ cells were pregated for the analysis. Cell debris, dead cells, and cell doublets were excluded from the analysis based on scatter signals and Viobility 405/520 Fixable Dye fluorescence. No FcR Blocking Reagent was used. The recommended titers of respective antibodies from different suppliers were used.

Flow cytometric comparison of different clones for IL-5. Human peripheral blood mononuclear cells (PBMCs) were stimulated with 20 ng/mL Phorbol 12-myristate 13-acetate (PMA) and 1 µg/ml Ionomycin for 1 hour, followed by an incubation with 1µg/mL Brefeldin A for 5 hours. Cells were first stained with Viobility™ Fixable Dye followed by a suitable counterstaining. Cells were then fixed and permeabilized followed by a staining with IL-5 antibodies. As a control, IL-5 antibody staining was omitted and cells were measured in the same channels. Flow cytometry was performed with the MACSQuant^® Analyzer. CD4+ cells were pregated for the analysis. Cell debris, dead cells, and cell doublets were excluded from the analysis based on scatter signals and Viobility 405/520 Fixable Dye fluorescence. No FcR Blocking Reagent was used. The recommended titers of respective antibodies from different suppliers were used.

Specifications for IL-5 Antibody, anti-human

Overview

Interleukin 5 (IL-5) is pre-dominantly secreted by CD4

⁺

T cells. It is involved in a range of allergic reactions and mediates immune reactions against parasites. IL-5 also acts on other cell types such as B cells.

The Anti-IL-5 antibody has been designed for intracellular staining of IL-5–producing cells. Cells can be stimulated for IL-5-production, for example, by polyclonal stimulation with mitogens. For induction of IL-5 production by antigen-specific T cells, cells are restimulated with respective antigen. IL-5 can be accumulated in the cells by addition of secretion inhibitors like brefeldin A. After fixation and permeabilization of the cell sample, IL-5–producing cells can be stained intracellularly with Anti-IL-5 antibodies. Staining of surface markers allows simultaneous flow cytometric analysis of subsets and activation status of the IL-5–producing cells. Magnetically enriched cells can be stained intracellularly for IL-5 production directly on the MACS

^®

Column. This procedure ensures higher sensitivity of detection and minimizes loss of cells during washing procedures. The protocol is useful for cytokine analysis of rare cells, for example, CD4

⁺

T cells in HIV patients, or other cell sources than peripheral blood mononuclear cells (PBMCs), e.g., bronchoalveolar lavages or synovial fluids.

Alternative names

EDF, TRF

Detailed product information

Technical specifications

Clone	JES1-39D10
Clonality	monoclonal
Isotype	rat IgG2aκ
Isotype control	Isotype Control Antibody, rat IgG2a
Host	rat
Type of antibody	Primary antibodies
Species	human, non-human primate
Cross-reactivity	rhesus monkey ( Macaca mulatta ) , cynomolgus monkey ( Macaca fascicularis )
Antigen	IL-5
Alternative names of antigen	EDF, TRF
Molecular mass of antigen [kDa]	13
Entrez Gene ID	3567
RRID	AB_2660013, AB_2660014, AB_2905289, AB_2905288, AB_2905291, AB_2905290, AB_615133

Resources for IL-5 Antibody, anti-human

Certificates

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link

to search for Certificates of Analysis (CoA) by lot number.

Applications

Extended validation