Please Note: This nucleic acid dye has been developed for the flow cytometry (intracellular) and immunofluorescence microscopy applications. Chemically characterized for consistency, researchers should determine the optimal concentration of this reagent for individual applications.
Staining of Live Cells for Viability Analysis by Flow Cytometry
1. Obtain a single cell suspension.
2. Resuspend cells at 1-2 × 10^6 cells/mL in BD Pharmingen™ Stain Buffer (FBS) (Cat. No. 554656) or 1× DPBS containing 5-20 nM BD Via-Probe™ Red.
a. The optimal concentration of BD Via-Probe™ Red for viability analysis may vary by cell type. Using high concentrations may result in the dye entering viable cells. Therefore, we recommend titrating the reagent for your cell type of interest in preliminary experiments.
b. Additionally, apoptotic cells may stain with variable amounts of probe. We recommend co-staining with a probe for apoptotic cells, eg, BD Horizon Annexin V (Cat. No. 563973), if further analysis of apoptotic cells is desired.
3. Incubate the cells and dye for 5 minutes at room temperature protected from light. No further cell washes are 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 DNA histograms. However, this reagent has also been successfully used for DNA content analysis with the BD Pharmingen™ Transcription Factor Buffer Set (Cat. No. 562574/562725) protocol.
3. Wash cells once with BD Pharmingen™ Stain Buffer (FBS).
4. Resuspend cells at 1-2 × 10^6 cells/mL in 0.1-1 μM BD Via-Probe™ Red in the presence of 0.1-0.5 mg/mL DNAse-free RNAse (eg, Sigma Aldrich, Cat. No. D6513) diluted in BD Pharmingen™ Stain Buffer (FBS) or 1× DPBS immediately before use.
a. The optimal cell density and concentration of BD Via-Probe™ Red for DNA content analysis may vary by cell type. Assay conditions should be optimized in preliminary experiments for best results.
5. Incubate cells for 5-15 minutes. No further cell washes are necessary prior to analysis.
6. Proceed to analysis by flow cytometry. Samples should be run at a low flow rate to achieve the best results. High flow rates may result in higher % CV for each cell cycle compartment in the DNA histogram.
Immunofluorescent Staining of Fixed Cells for Nuclear Visualization
1. Fix and permeabilize cells as desired.
2. Dilute BD Via-Probe™ Red solution to 0.1-1 μM in 1× DPBS with 0.1-0.5 mg/mL DNase-free RNase (eg, Sigma Aldrich, Cat. No. D6513) immediately prior to use.
3. Add BD Via-Probe™ Red solution to each sample at least 15 minutes before analysis.
4. Proceed to fluorescence microscopy and image analysis.
