BD, 564907, BD Pharmingen™ DAPI Solution

Application: Bioimaging, Flow cytometry, Immunofluorescence (Tested During Development)
Concentration: 1.0 mg/ml
RRID: AB_2869624
Regulatory Status: RUO
Preparation And Storage: Preparation And Storage Avoid multiple freeze-thaws of product. The product should be kept undiluted at -20°C for long term storage, and it may be kept undiluted at 4°C for short term storage.
Recommended Assay Procedures: Recommended Assay Procedures Staining of Live Cells for Viability Analysis by Flow Cytometry 1. Obtain a single cell suspension. 2. Resuspend cells in BD Pharmingen™ Stain Buffer (FBS) (Cat. No. 554656) or 1× Dulbecco's Phosphate Buffered Saline (DPBS) containing 0.05-0.2 μg/mL DAPI. a. The optimal concentration of DAPI for viability analysis may vary by cell type. We recommend titrating the reagent for your cell type of interest in early experiments. b. Additionally, apoptotic cells may stain with variable amounts of DAPI. We recommend co-staining with BD Pharmingen™ FITC Annexin V (Cat. No. 556419) if further analysis of apoptotic cells is desired. 3. Incubate 5 minutes at room temperature. No wash is necessary prior to analysis. 4. Proceed to analysis by flow cytometry. Staining of Fixed Cells for DNA Content Analysis by Flow Cytometry 1. Obtain a single cell suspension. 2. Treat cells on ice for 30 minutes with 70-80% ice-cold ethanol. a. Ethanol fixation typically provides the most resolved histograms. However, this reagent has also been successfully used for DNA content analysis with the Transcription Factor Buffer Set (Cat. No. 562574/ 562725) or BD Cytofix™ Fixation Buffer (Cat. No. 554655) and BD Phosflow™ Perm Buffer III (Cat. No. 558050) protocol. 3. Wash cells once with BD Pharmingen™ Stain Buffer (FBS). 4. Dilute DAPI solution to 0.5-1 μg/mL in Stain Buffer (FBS) or 1× DPBS immediately prior to use. 5. Stain cells for 5-15 minutes at a cell density of 1 - 2 x 10^6 cells/mL. No further wash is necessary prior to analysis. a. The optimal cell density and concentration of DAPI for DNA content analysis may vary by cell type. Assay conditions should be optimized in early experiments for best results. 6. Proceed to analysis by flow cytometry. Immunofluorescent Staining of Fixed Cells for Nuclear Visualization 1. Fix and permeabilize cells as desired. 2. Dilute DAPI solution to 1 µg/ml in 1× DPBS immediately prior to use. 3. Add DAPI solution to each sample at least 15 minutes before analysis. 4. Proceed to imaging. Note: This reagent has been developed and certified for the Bioimaging application. However, routine Bioimaging testing is not performed on every lot.
Product Notices: Product Notices FlowJo is a trademark of Tree Star Inc. Alexa Fluor® is a registered trademark of Molecular Probes, Inc., Eugene, OR. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.