BD Pharmingen™ Blue Live Cell Caspase Probe 565519

Application: Flow cytometry, Intracellular staining (flow cytometry) (Tested During Development)
RRID: AB_2869680
Regulatory Status: RUO
Preparation And Storage: Preparation And Storage Store at -20°C, protected from exposure to light.
Recommended Assay Procedures: Recommended Assay Procedures Note: Before staining, confirm that your flow cytometer is capable of exciting the fluorochrome and discriminating the resulting fluorescence. Preparation Bring lyophilized probe and 50 μl of fresh cell culture-grade Dimethyl Sulfoxide (DMSO; e.g. Sigma D2650) to room temperature. Add 50 μl of DMSO and vortex solution well. Inspect the solution and repeat vortex until the stock dye has fully dissolved. This is the Stock Solution. Storage Upon arrival, store the dry probe desiccated and protected from light at -20°C until use. After reconstitution with DMSO, store the Stock Solution at -20°C in small aliquots. The Stock Solution is stable for at least 3 months post reconstitution with DMSO. Cytometry Requirements Blue laser-equipped Flow Cytometers (eg, BD FACSCanto™ II, BD LSRFortessa™, BD™ LSR II, and BD Accuri™ C6) can be used. This dye can be read out of filters commonly used for FITC (eg, 530/30 nm). Fluorescence compensation is best achieved using a sample of the cells of interest stained with the dye. When designing multicolor panels, we recommend titrating the dye and using the lowest possible concentration that provides adequate resolution of positive and negative populations for the cell type of interest to reduce spillover. Procedure Labeling of Cells with BD Pharmingen™ Blue Live Cell Caspase Probe 1. Count cells to determine cell density. Adjust cell density to 1-2 × 10^6 cells/ml in fresh, pre-warmed cell culture media. a. Adherent cells may be stained either in situ or in suspension after removal from the growth substrate. Cells should be 70% confluent or less when assayed. b. Note: One "test" is defined as a 500 μL aliquot of cells at 1-2 × 10^6 cells/ml. 2. Add 2 μL of Stock Solution per 1 mL of cell suspension or culture media. For example, for a 500 μL aliquot of cells, add 1 μL of the Stock Solution. Mix thoroughly. a. In some cases, it may be useful to titrate the probe, as different cell types and different applications can result in variability in staining intensities. 3. Incubate samples for 30-60 minutes at 37°C protected from light. 4. Wash cells once with fresh, pre-warmed media. 5. Resuspend cells in fresh, pre-warmed media and incubate cells for an additional 15-60 minutes at 37°C protected from light to allow unbound probe to diffuse out of the cells. 6. Pellet cells once more by centrifugation and remove the supernatant. 7. Resuspend the cells in 1× Dulbecco's Phosphate Buffered Saline (DPBS) or equivalent. a. Note: Dead cells bind variable amounts of probe. We recommend co-staining with an additional fluorescent probe such as BD Via-Probe™ Cell Viability Solution (Cat. No. 555816) in order to distinguish dead cells from live or apoptotic cells. 8. Analyze samples by flow cytometry. Notes: 1. Each user should determine the optimal concentrations of reagents, cells, and conditions for the assay of interest. We recommend titrating the reagent in early experiments to obtain optimal results. 2. BD Pharmingen™ Blue Live Cell Caspase Probe is compatible with fixation and permeabilization protocols such as those used for BD Phosflow™ staining (eg, Cat. No. 558050, BD Phosflow™ Perm Buffer III) or intracellular cytokine staining (eg, Cat. No. 554714, BD Cytofix/Cytoperm™ Fixation/Permeabilization Solution Kit). However, please note that resolution of positive and negative populations is reduced in fixed and permeabilized cells as compared to live cells. Where possible, it is recommended that samples be analyzed live.
Product Notices: Product Notices Since applications vary, each investigator should titrate the reagent to obtain optimal results. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors. Before staining with this reagent, please confirm that your flow cytometer is capable of exciting the fluorochrome and discriminating the resulting fluorescence. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.