BD Pharmingen™ PE Rat IgG1, κ Isotype Control

Immunofluorescent Staining for Intracellular Cytokines: The PE-R3-34 immunoglobulin is a suitable rat IgG1 isotype control for assessing the level of background staining on paraformaldehyde-fixed/saponin-permeabilized mouse or human cells for flow cytometric analysis. This 100 Test Size formulation of the PE-conjugated R3-34 antibody has been pre-titrated to assure effective results as an Ig isotype control for our Test Sized PE-conjugated Rat IgG1 antibodies using 20µl/1 x 10^6 cells. The intracellular cytokine staining techinque and use of blocking controls are described in detail by C. Prussin and D. Metcalfe. For specific methodology, please visit the protocols section or chapter on intracellular staining in the Immune Function Handbook, both of which are posted on our web site, www.bdbiosciences.com.

Important Note: This pre-titered antibody solution does not contain a cell permeabilization agent. It is necessary to include a cell permeabilization agent when using the pre-titered antibody solution to stain fixed and permeabilized cells. BD Perm/Wash™ Buffer (Cat. No. 554723) contains the permeabilization agent saponin and is useful for this purpose as described in the Usage section below.

Usage

1. Resuspend 1 x 10^6 fixed and permeabilized cells in 20 µl of the pre-titered antibody solution and 30 µl of 1 x BD Perm/Wash™ Buffer (Cat. No. 554723).

2. Incubate the cell suspension for 15 minutes (at RT or 4°C).

3. Wash twice in 100 µl of 1 x BD Perm/Wash Buffer (Cat. No. 554723).

Bead-based compensation or unmixing controls, such as BD® CompBeads or BD™ SpectraComp™, can be used as surrogates to assess fluorescence spillover when bound to fluorochrome-conjugated antibodies. Although these beads have spectral properties similar to cells, variations in spectral emission may occur, resulting in differing spillover values compared to biological controls. Therefore, it is considered best practice to compare the spillover obtained from cells and bead-based controls when using BD® CompBeads or BD™ SpectraComp™ for the first time, to ensure they are appropriate for the intended application.

1. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
2. Source of all serum proteins is from USDA inspected abattoirs located in the United States.
3. 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.
4. 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).
5. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
6. An isotype control should be used at the same concentration as the antibody of interest.
7. Please refer to http://regdocs.bd.com to access safety data sheets (SDS).
8. For U.S. patents that may apply, see bd.com/patents.