Clone:
JES1-39D10
Type of antibody:
Primary antibodies
Isotype:
rat IgG2aκ
Applications:
ICFC
Alternative names:
EDF, TRF

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 clonesOverlap 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.
View details
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.
View details
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.
View details
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.
View details
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.
View details
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

CloneJES1-39D10
Clonalitymonoclonal
Isotyperat IgG2aκ
Isotype controlIsotype Control Antibody, rat IgG2a
Hostrat
Type of antibodyPrimary antibodies
Specieshuman, non-human primate
Cross-reactivity
rhesus monkey (
Macaca mulatta
)
,
cynomolgus monkey (
Macaca fascicularis
)
AntigenIL-5
Alternative names of antigenEDF, TRF
Molecular mass of antigen [kDa]13
Entrez Gene ID3567
RRIDAB_2660013, AB_2660014, AB_615133

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