BD, 565879, BD Pharmingen™ Indo-1 AM

Application: Flow cytometry (Tested During Development)
RRID: AB_2869725
Regulatory Status: RUO
Recommended Assay Procedures: Recommended Assay Procedures Preparation Bring Indo-1 AM dye powder and anhydrous Dimethyl Sulfoxide to room temperature. Add 10 μL of DMSO to dye powder and vortex solution well. Inspect the solution and repeat vortex until the stock dye has fully dissolved. This yields a 5 mM stock solution. Storage Upon arrival, store the dry dye desiccated and protected from light at -20°C until use. We recommend a fresh vial of dye be used for each experiment and that reconstituted dye be discarded after use. However, if stock solutions are to be kept for use, they should be stored desiccated and protected from light at -20°C and used within one week of reconstitution. Cytometry Requirements UV (355 nm) laser-equipped flow cytometers (eg, BD LSRFortessa™, BD LSRFortessa™ X-20, or BD™ LSR II) can be used. This dye is ratiometric and requires filter sets around 400 nm (calcium bound) and 500 nm (calcium free). Filter sets for BUV395 (eg, 379/28) and BUV496 (eg, 515/30) in conjunction with a 450 nm dichroic mirror between the filter sets work well. Ratiometric changes in calcium concentration are usually calculated as the ratio of emission in the low wavelength filter to the emission in the high wavelength filter. Fluorescence compensation is best achieved using a sample of the cells of interest stained with the dye. When designing multicolor panels, please be aware of spillover into the BD Horizon™ BV421 and BD Horizon BV510 channels. Panels should be optimized to take this spillover into account. Procedure BD Pharmingen™ Indo-1 AM Labeling of Cells 1. Prepare a single cell suspension at 1 × 10e6 cells/mL in physiologic loading buffer of choice. a. If serum is used in the loading buffer, it should be heat inactivated in order to prevent residual serum esterase activity from cleaving AM moieties on the dye prior to entry into cells. 2. Add dye stock solution for a final staining concentration of 1-10 μM and vortex immediately. a. We recommend using the lowest dye concentration that still yields sufficient signal in order to avoid dye toxicity, compartmentalization, and calcium buffering. 3. Incubate 15-60 minutes at 37°C. a. It is reported that dye compartmentalization is less significant at lower loading temperatures. In this case, it may be advantageous to load cells at room temperature if dye compartmentalization is significant for the cell type of interest. 4. Wash once and resuspend in analysis buffer of choice. a. For some cell types, it may be advantageous to permit cells to recover for another 30-60 minutes to allow complete de-esterification of AM moieties. In this case, cells should be incubated in physiologic buffer of choice or complete medium, washed once more, and then resuspended in analysis buffer of choice. 5. Proceed to flow cytometry. a. BD FACSDiva™ users may create a ratiometric parameter before acquisition on the Ratio tab of the Cytometer menu. This parameter cannot be edited during acquisition. Therefore, it may be useful to create controls (eg, a control sample to be stimulated with ionophore A23187 or ionomycin) to adjust the ratiometric parameter scaling ahead of time in order to ensure that the ratiometric values will be on scale. b. Alternatively, data may be acquired for each non-ratiometric parameter individually and then the ratiometric parameter may be calculated from the raw data in FlowJo™ (FlowJo, LLC) as a Derived Parameter. Note: Before staining with this reagent, please confirm that your flow cytometer is capable of exciting the fluorochrome and discriminating the resulting fluorescence.
Product Notices: Product Notices Since applications vary, each investigator should titrate the reagent to obtain optimal results. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.