The monoclonal antibody was purified from tissue culture supernatant or ascites by affinity chromatography. The antibody was conjugated with R-PE under optimum conditions, and unconjugated antibody and free PE were removed. Store undiluted at 4°C and protected from prolonged exposure to light. Do not freeze.
An isotype control should be used at the same concentration as the antibody of interest (e.g., ≤ 0.5 µg/million cells for flow cytometry).
The intracellular cytokine staining technique and use of blocking controls are described in detail by C. Prussin and D. Metcalfe. For specific methodology, please visit our web site, www.bdbiosciences.com, and refer to the protocols section or the chapter on intracellular staining in the Immune Function Handbook.
Since applications vary, each investigator should titrate the reagent to obtain optimal results.
Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
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.
The R35-95 hybridoma was generated by hybridization of Y3 myeloma cells with spleen cells from LOU rats immunized with mouse immunoglobulins. The R35-95 hybridoma produces rat IgG2a, κ immunoglobulin that has no measurable reactivity with mouse immunoglobulins. The R35-95 immunoglobulin was selected as an isotype control following screening for low background binding on a variety of mouse and human tissues.
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R-Phycoerythrin (PE), is part of the BD family of Phycobiliprotein dyes. This fluorochrome is a multimeric fluorescent phycobiliprotein with excitation maximum (Ex Max) of 496 nm and 566 nm and an emission maximum (Em Max) at 576 nm. PE is designed to be excited by the Blue (488 nm), Green (532 nm) and Yellow-Green (561 nm) lasers and detected using an optical filter centered near 575 nm (e.g., a 575/26-nm bandpass filter). As PE is excited by multiple lasers, this can result in cross-laser excitation and fluorescence spillover on instruments with various combinations of Blue, Green, and Yellow-Green lasers. Please ensure that your instrument’s configurations (lasers and optical filters) are appropriate for this dye.
Yellow-Green 488 nm, 532 nm, 561 nm
496 nm, 566 nm
Citations & References
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).