Alexa Fluor® 488 Rat IgG1, κ Isotype Control
Clone R3-34 (RUO)
- Brand BD Pharmingen™
- Vol. Per Test 5 µl
- Isotype Rat IgG1, κ
Intracellular staining (flow cytometry), Isotype control (Routinely Tested)
- Immunogen Mouse immunoglobulin
- Storage Buffer Aqueous buffered solution containing BSA and ≤0.09% sodium azide.
- Regulatory Status RUO
Regulatory Status Legend
Following immunization of a rat with mouse immunoglobulin (Ig), the Ig from the R3-34 hybridoma was identified as a non-reactive clone. The R3-34 immunoglobulin was selected as an Ig isotype control following screening for low background staining on a variety of mouse and human cells and tissues.
This antibody is routinely tested by flow cytometric analysis. Other applications were tested at BD Biosciences Pharmingen during antibody development only or reported in the literature.
Alexa Fluor® conjugates are highly photostable and remain fluorescent over a broad pH range. The excitation and emission maxima are nearly identical to those of FITC. However, Alexa Fluor® 488 tends to be brighter and more optimal for intracellular applications. Due to nearly identical excitation and emission properties but different spillover characteristics, FITC and Alexa Fluor® 488 cannot be used simultaneously. Alexa Fluor® 488 exhibits extraordinary photostability, which makes it highly suitable for fluorescence microscopy.
Preparation and Storage
The monoclonal antibody was purified from tissue culture supernatant or ascites by affinity chromatography.
The antibody was conjugated to Alexa Fluor® 488 under optimum conditions, and unreacted Alexa Fluor® 488 was removed.
Store undiluted at 4°C and protected from prolonged exposure to light. Do not freeze.
- 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).
- Since applications vary, each investigator should titrate the reagent to obtain optimal results.
- Please refer to www.bdbiosciences.com/pharmingen/protocols for technical protocols.
- For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
- Alexa Fluor® 488 fluorochrome emission is collected at the same instrument settings as for fluorescein isothiocyanate (FITC).
- 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.
- Source of all serum proteins is from USDA inspected abattoirs located in the United States.
- 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.
Recommended Assay Procedure:
Immunofluorescent Staining for Intracellular Cytokines: The FITC-, PE-, APC-, PE-Cy7-, Alexa Fluor® 488-, and Alexa Fluor® 647-conjugated-R3-34 immunoglobulin (Cat. No. 554684, 554685, 557856, 557865, 554686, and 557645) is a suitable rat IgG1 κ isotype control for assessing the level of background staining on paraformaldehyde-fixed/saponin-permeabilized human cells for flow cytometric analysis. Use at 5 µl/test. 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 website, www.bdbiosciences.com, and go to the protocols section or the chapter on intracellular staining in the Immune Function Handbook.
An isotype control should be used at the same concentration as the antibody of interest.