BD Pharmingen™ Alexa Fluor® 647 Mouse Anti-Rat IL-10
Clone A5-4 (RUO)
Flow cytometric analysis of IL-10 expression by stimulated CD4+ rat splenocytes. Purified splenic CD4+ cells from Lewis rats were stimulated with plate-bound Purified NA/LE Mouse Anti-Rat CD3 (10 μg/ml; Cat. No. 554829) and soluble Purified NA/LE Mouse Anti-Rat CD28 (2 μg/ml; Cat. No. 554993) for 2 days in complete medium with Recombinant Rat IL-2 (10 ng/ml; Cat. No. 555106) and Recombinant Rat IL-4 (40 ng/ml; Cat. No 555107). Cells were harvested and cultured for 3 days in the presence of IL-2 (10 ng/ml) and IL-4 (20 ng/ml). This was followed by a 6 hour stimulation with Phorbol 12-Myristate 13-Acetate (PMA; 5 ng/ml; Sigma, Cat. #P-8139) and Ionomycin (500 ng/ml; Sigma, Cat. #I-0634) in the presence of BD GolgiPlug™ Protein Transport Inhibitor (Containing Brefeldin A) (2 μM; Cat. No. 555029). The cells were then fixed with BD Cytofix™ Fixation Buffer (Cat. No. 554655), and permeabilized with BD Perm/Wash™ Buffer (Cat. No. 554723), and subsequently stained with either Alexa Fluor® 647 IgG1, κ Isotype Control (Cat. No. 557732, Left Panel) or Alexa Fluor® 647 Mouse anti-Rat IL-10 antibody (Middle and Right Panels, Cat. No. 562156) by using BD Biosciences Intracellular Cytokine Staining Protocol. To demonstrate the specificity of staining, the binding of the Alexa Fluor® 647 Mouse anti-Rat IL-10 antibody was blocked by preincubation of cells with the Purified Mouse anti-Rat IL-10 antibody (Clone A5-4; Right Panel) prior to staining. Flow cytometric fluorescence dot blots showing the expression of IL-10 (or Ig Isotype background staining) versus cellular autofluorescence (measured in the FL2 channel) were derived from gated events with the forward and side light-scatter characteristics of intact splenocytes. Flow cytometry was performed using a BD™ LSR II Flow Cytometer System.
Flow cytometric analysis of IL-10 expression by stimulated CD4+ rat splenocytes. Purified splenic CD4+ cells from Lewis rats were stimulated with plate-bound Purified NA/LE Mouse Anti-Rat CD3 (10 μg/ml; Cat. No. 554829) and soluble Purified NA/LE Mouse Anti-Rat CD28 (2 μg/ml; Cat. No. 554993) for 2 days in complete medium with Recombinant Rat IL-2 (10 ng/ml; Cat. No. 555106) and Recombinant Rat IL-4 (40 ng/ml; Cat. No 555107). Cells were harvested and cultured for 3 days in the presence of IL-2 (10 ng/ml) and IL-4 (20 ng/ml). This was followed by a 6 hour stimulation with Phorbol 12-Myristate 13-Acetate (PMA; 5 ng/ml; Sigma, Cat. #P-8139) and Ionomycin (500 ng/ml; Sigma, Cat. #I-0634) in the presence of BD GolgiPlug™ Protein Transport Inhibitor (Containing Brefeldin A) (2 μM; Cat. No. 555029). The cells were then fixed with BD Cytofix™ Fixation Buffer (Cat. No. 554655), and permeabilized with BD Perm/Wash™ Buffer (Cat. No. 554723), and subsequently stained with either Alexa Fluor® 647 IgG1, κ Isotype Control (Cat. No. 557732, Left Panel) or Alexa Fluor® 647 Mouse anti-Rat IL-10 antibody (Middle and Right Panels, Cat. No. 562156) by using BD Biosciences Intracellular Cytokine Staining Protocol. To demonstrate the specificity of staining, the binding of the Alexa Fluor® 647 Mouse anti-Rat IL-10 antibody was blocked by preincubation of cells with the Purified Mouse anti-Rat IL-10 antibody (Clone A5-4; Right Panel) prior to staining. Flow cytometric fluorescence dot blots showing the expression of IL-10 (or Ig Isotype background staining) versus cellular autofluorescence (measured in the FL2 channel) were derived from gated events with the forward and side light-scatter characteristics of intact splenocytes. Flow cytometry was performed using a BD™ LSR II Flow Cytometer System.
Product Details
BD Pharmingen™
Il10; Interleukin-10; CSIF; Cytokine synthesis inhibitory factor
Rat (QC Testing)
Mouse IgG2b, κ
Recombinant Rat IL-10
Intracellular staining (flow cytometry) (Routinely Tested)
0.2 mg/ml
AB_11153120
Aqueous buffered solution containing ≤0.09% sodium azide.
RUO
Preparation And Storage
Store undiluted at 4°C and protected from prolonged exposure to light. Do not freeze. The monoclonal antibody was purified from tissue culture supernatant or ascites by affinity chromatography. The antibody was conjugated to Alexa Fluor® 647 under optimum conditions, and unreacted Alexa Fluor® 647 was removed.
Product Notices
- Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
- Since applications vary, each investigator should titrate the reagent to obtain optimal results.
- The Alexa Fluor®, Pacific Blue™, and Cascade Blue® dye antibody conjugates in this product are sold under license from Molecular Probes, Inc. for research use only, excluding use in combination with microarrays, or as analyte specific reagents. The Alexa Fluor® dyes (except for Alexa Fluor® 430), Pacific Blue™ dye, and Cascade Blue® dye are covered by pending and issued patents.
- Alexa Fluor® is a registered trademark of Molecular Probes, Inc., Eugene, OR.
- Alexa Fluor® 647 fluorochrome emission is collected at the same instrument settings as for allophycocyanin (APC).
- Caution: Sodium azide yields highly toxic hydrazoic acid under acidic conditions. Dilute azide compounds in running water before discarding to avoid accumulation of potentially explosive deposits in plumbing.
- For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
- An isotype control should be used at the same concentration as the antibody of interest.
Companion Products
Stain Buffer (FBS) RUO
Size
500 mL
Cat No.
554656
Fixation Buffer RUO
Size
100 mL
Cat No.
554655
Perm/Wash Buffer RUO
Size
100 mL
Cat No.
554723
Alexa Fluor® 647 Mouse IgG1 κ Isotype Control RUO
Size
100 Tests
Cat No.
557732
Protein Transport Inhibitor (Containing Brefeldin A) RUO
Size
1 mL
Cat No.
555029
Purified NA/LE Mouse Anti-Rat CD3 RUO
Size
0.5 mg
Cat No.
554829
562156 Rev. 1
Antibody Details
A5-4
The A5-4 monoclonal antibody specifically binds to rat interleukin-10 (IL-10). The immunogen used to generate the A5-4 hybridoma was recombinant rat IL-10 protein.
562156 Rev. 1
Format Details
Alexa Fluor™ 647
Alexa Fluor™ 647 Dye is part of the BD red family of dyes. This is a small organic fluorochrome with an excitation maximum (Ex Max) at 653-nm and an emission maximum (Em Max) at 669-nm. Alexa Fluor 647 is designed to be excited by the Red laser (627-640 nm) and detected using an optical filter centered near 520-nm (e.g., a 660/20 nm bandpass filter). Please ensure that your instrument’s configurations (lasers and optical filters) are appropriate for this dye.
Alexa Fluor™ 647
Red 627-640 nm
653 nm
669 nm
562156 Rev.1
Citations & References
View product citations for antibody "562156" on CiteAb
Development References (3)
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Feng L, Tang WW, Chang JC, Wilson CB. Molecular cloning of rat cytokine synthesis inhibitory factor (IL-10) cDNA and expression in spleen and macrophages. Biochem Biophys Res Commun. 1993; 192(2):452-458. (Immunogen). View Reference
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Prakken BJ, Wendling U, van der Zee R, Rutten VP, Kuis W, van Eden W.. Induction of IL-10 and inhibition of experimental arthritis are specific features of microbial heat shock proteins that are absent for other evolutionarily conserved immunodominant proteins. J Immunol. 2001; 167(8):4147-4153. (Clone-specific: IC/FCM Block). View Reference
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Prussin C, Metcalfe DD. Detection of intracytoplasmic cytokine using flow cytometry and directly conjugated anti-cytokine antibodies. J Immunol Methods. 1995; 188(1):117-128. (Methodology: Flow cytometry, IC/FCM Block). View Reference
562156 Rev. 1
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Comparisons, where applicable, are made against older BD Technology, manual methods or are general performance claims. Comparisons are not made against non-BD technologies, unless otherwise noted.
For Research Use Only. Not for use in diagnostic or therapeutic procedures.