BD Pharmingen™ Three-color Fluorescent Ig Isotype Cocktail with Human CD4+ PerCP-Cy™5.5

The multicolor Ig isotype control cocktail is designed to control for nonspecific staining by fluorescent antibodies directed against cellular antigens found in target CD4+ T cells that have been fixed with BD Cytofix™ Fixation Buffer (Cat. No. 554655) and permeabilized with BD Perm/Wash™ Buffer (Cat. No. 554723), or the BD Cytofix/Cytoperm™ Fixation/Permeabilization Solution Kit (Cat. No. 554714). Nonspecific staining by fluorescent antibodies may be due to nonspecific immunoglobulin and/or fluorochrome- mediated binding to cellular molecules including Fc receptors for immunoglobulin. In addition, this multicolor Ig isotype cocktail can be used for negative control staining for other multicolor fluorescent antibody cocktails that contains PerCP-Cy ™ 5.5 anti-Human CD4 mixed with FITC- and PE-conjugated Mouse IgG1, κ and APC-Rat IgG1, κ antibo dies. As such, the cocktail can provide negative controls for the immunofluorescent staining with fluorescent antibodies specific for other intracellular markers (eg, cytokines, chemokines, and/or transcription factors) expressed by CD4+ T cells that are prepared using BD Cytofix™ Fixation buffer and BD Perm/Wash™ Perm/Wash Buffer.

See the protocol for stimulating, fixing, permeabilizing, and staining cells provided in the Technical Data Sheet for the Human Th1/Th2/Th17 Phenotyping Kit (Cat. No. 560751).

1. This reagent has been pre-diluted for use at the recommended Volume per Test. We typically use 1 × 10^6 cells in a 100-µl experimental sample (a test).
2. 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.
3. Source of all serum proteins is from USDA inspected abattoirs located in the United States.
4. This APC-conjugated reagent can be used in any flow cytometer equipped with a dye, HeNe, or red diode laser.
5. Please observe the following precautions: Absorption of visible light can significantly alter the energy transfer occurring in any tandem fluorochrome conjugate; therefore, we recommend that special precautions be taken (such as wrapping vials, tubes, or racks in aluminum foil) to prevent exposure of conjugated reagents, including cells stained with those reagents, to room illumination.
6. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
7. PerCP-Cy5.5–labelled antibodies can be used with FITC- and R-PE–labelled reagents in single-laser flow cytometers with no significant spectral overlap of PerCP-Cy5.5, FITC, and R-PE fluorescence.
8. PerCP-Cy5.5 is optimized for use with a single argon ion laser emitting 488-nm light. Because of the broad absorption spectrum of the tandem fluorochrome, extra care must be taken when using dual-laser cytometers, which may directly excite both PerCP and Cy5.5™. We recommend the use of cross-beam compensation during data acquisition or software compensation during data analysis.
9. This product is subject to proprietary rights of Amersham Biosciences Corp. and Carnegie Mellon University and made and sold under license from Amersham Biosciences Corp. This product is licensed for sale only for research. It is not licensed for any other use. If you require a commercial license to use this product and do not have one return this material, unopened to BD Biosciences, 10975 Torreyana Rd, San Diego, CA 92121 and any money paid for the material will be refunded.
10. Cy is a trademark of Amersham Biosciences Limited. This conjugated product is sold under license to the following patents: US Patent Nos. 5,486,616; 5,569,587; 5,569,766; 5,627,027.
11. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.