BD, 564902, BD Pharmingen™ DRAQ5™

Application: Bioimaging, Immunofluorescence, Intracellular staining (flow cytometry) (Tested During Development)
Concentration: 5 mM
RRID: AB_2869620
Regulatory Status: RUO
Preparation And Storage: Preparation And Storage Store undiluted at 4°C and protected from prolonged exposure to light. Do not freeze.
Recommended Assay Procedures: Recommended Assay Procedures Staining of Live Cells for DNA Content Analysis by Flow Cytometry 1. Obtain a single cell suspension. 2. Resuspend cells at 0.5 x 10^6 cells/mL or less in complete medium or other azide-free buffer containing 20 μM DRAQ5™. 3. Incubate at 37°C for 5-15 minutes. a. The optimal cell density, concentration of DRAQ5™, and stain time for DNA content analysis may vary by cell type. Assay conditions should be optimized in early experiments for best results. 4. Pellet cells by centrifugation and aspirate medium or other azide-free buffer containing DRAQ5™. 5. Resuspend cells in BD Pharmingen™ Stain Buffer (FBS) (Cat. No. 554656) or 1× PBS and immediately proceed to analysis by flow cytometry. Cells may also be analyzed without washing, but this may decrease DNA content histogram resolution. 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) or BD Cytofix™ Fixation Buffer (Cat. No. 554655) and BD Phosflow™ Perm III (Cat. No. 558050) protocol. 3. Wash cells once with BD Pharmingen™ Stain Buffer (FBS). 4. Dilute DRAQ5™ solution to 20 μM in 1× Dulbecco's Phosphate Buffered Saline (DPBS) or other azide-free buffer immediately prior to use. 5. Stain cells for 5-15 minutes at a cell density of 0.5 x 10^6 cells/mL or less. No further wash is necessary prior to analysis. a. The optimal cell density and concentration of DRAQ5™ 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 Live Cells for Nuclear Visualization 1. Dilute DRAQ5™ solution to 5-20 μM in complete medium or other azide-free buffer immediately prior to use. 2. Add DRAQ5™ solution to samples and incubate at 37°C for 5-30 minutes. The stain time required is cell type dependent. 3. Remove DRAQ5™ solution from cells at the end of the incubation period and add BD Pharmingen™ Stain Buffer (FBS) (Cat. No. 554656) or 1× DPBS. Cells may also be analyzed without washing, but this may increase background from unbound dye. 4. Proceed to imaging. We recommend using a 715LP or longer wavelength filter, though the dye is well-detected in filters typically used to detect Alexa Fluor® 647 (eg, 660/20 or 692/40 nm). Note that dsDNA-bound dye will fluoresce brightly in the nucleus and unbound dye may fluoresce dimly in the cytoplasm, allowing segmentation of the cytoplasmic and nuclear compartments. Immunofluorescent Staining of Fixed Cells for Nuclear Visualization 1. Fix and permeabilize cells as desired. 2. Dilute DRAQ5™ solution to 5-20 μM in 1× DPBS or other azide-free buffer immediately prior to use. 3. Add DRAQ5™solution to each well at least 5 minutes before analysis. 4. Proceed to imaging. We recommend using a 715LP or longer wavelength filter, though the dye is well-detected in filters typically used to detect Alexa Fluor® 647 (eg, 660/20 or 692/40 nm). Note that dsDNA-bound dye will fluoresce brightly in the nucleus and unbound dye may fluoresce dimly in the cytoplasm, allowing segmentation of the cytoplasmic and nuclear compartments. Note: This reagent has been developed and certified for the Bioimaging application. However, a routine Bioimaging test is not performed on every lot. Warning: DRAQ5™ contains < 1% 1,5-BIS{[2-(DIMETHYLAMINO)ETHYL]AMINO}-4,8-DIHYDROXYANTHRACENE-9,10-DIONE Hazard statements Causes skin irritation. Causes serious eye irritation. May cause respiratory irritation. Precautionary statements Wear protective gloves/protective clothing/eye protection/face protection. Wash thoroughly after handling. IF IN EYES: Rinse cautiously with water for several minutes. Remove contact lenses, if present and easy to do. Continue rinsing. If eye irritation persists: Get medical advice/attention. Take off contaminated clothing and wash before reuse. Call a POISON CENTER or doctor/physician if you feel unwell. If skin irritation occurs: Get medical advice/attention.IF ON SKIN: Wash with plenty of water Avoid breathing dust/fume/gas/mist/vapors/spray. Use only outdoors or in a well-ventilated area. IF INHALED: Remove person to fresh air and keep comfortable for breathing. Store in a well-ventilated place. Keep container tightly closed.  Store locked up. Dispose of contents/container to an appropriate treatment and disposal facility in accordance with applicable laws and regulations, and product characteristics at time of disposal.