Indo 1 AM — 565879

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1 BD Pharmingen™ Technical Data Sheet Indo-1 AM Product Information 565879 Material Number: 500 μg Size: 51-9011907 Component: Indo-1 AM Description: 50ug (10 ea) Size: Description BD Pharmingen™ Indo-1 AM is a cell-permeant dye that can be used to measure intracellular ionized calcium concentration, [Ca2+]i. The hydrophobic acetomethoxy (AM) moiety of the dye allows passage across the cell membrane into viable cells. Once inside, intracellular esterases cleave the AM groups, leaving Indo-1 trapped within the cell and free to bind intracellular calcium. The Kd for Ca2+ is approximately 250 nM at ionic strengths near 0.10-0.15 M. BD Pharmingen™ Indo-1 AM is a ratiometric dye: when calcium is bound to the dye, the maximal excitation wavelength shifts from 349 to 331 nm and the maximal emission wavelength shifts from 485 nm to 410 nm. Thus, with UV excitation (eg. 355 nm), the ratio of emission around 410 nm to the emission around 485 nm can be used to measure intracellular ionized calcium concentration. Intracellular calcium response of Jurkat cells to stimulation with A23187. Jurkat cells were stained with 5 μ M BD Pharmingen™ Indo-1 AM (Cat. No. 565879) in DPBS with calcium and magnesium for 30 minutes at 37°C. Cells were pelleted once and further incubated in complete medium for 30 minutes to allow complete hydrolysis of AM moieties. Cells were then pelleted and resuspended in DPBS with calcium and magnesium and analyzed using an BD LSRFortessa™ Cell Analyzer System with a 355 nm laser, 379/28 (calcium bound) and 515/30 (calcium free) bandpass filters, and a 450 dichroic mirror between. After 30 seconds of acquisition, the sample tube was briefly removed from the cytometer to facilitate addition of 1 M calcium ionophore A23187 (Sigma Aldrich, Cat. No. C7522). Ratio: Bound/Free was μ calculated as the mean fluorescence intensity from the calcium bound channel (Left Plot) divided by the mean fluorescence intensity of the calcium free channel (Middle Plot), and FlowJo Kinetic analyzer (FlowJo, LLC) was used to plot the average ratio versus time (Right Plot). Jurkat cells show an increase in intracellular calcium concentration with addition of calcium ionophore A23187. Application Notes Application Flow cytometry Tested During Development Recommended Assay Procedure: Preparation μ L of DMSO to dye powder and vortex solution Bring Indo-1 AM dye powder and anhydrous Dimethyl Sulfoxide to room temperature. Add 10 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. Page 1 of 2 565879 Rev. 1

2 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. Add dye stock solution for a final staining concentration of 1-10 μ M and vortex immediately. 2. a. We recommend using the lowest dye concentration that still yields sufficient signal in order to avoid dye toxicity, compartmentalization, and calcium buffering. Incubate 15-60 minutes at 37°C. 3. It is reported that dye compartmentalization is less significant at lower loading temperatures. In this case, it may be a. 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. Proceed to flow cytometry. 5. 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 1. Since applications vary, each investigator should titrate the reagent to obtain optimal results. Please refer to www.bdbiosciences.com/pharmingen/protocols for technical protocols. 2. 3. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors. References Bruton JD, Cheng AJ, Westerblad H. Methods to detect Ca(2+) in living cells. Adv Exp Med Biol. 2012; 740:27-43. (Methodology) Grynkiewicz G, Poenie M, Tsien RY. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem. 1985; 260(6):3440-50. (Methodology) Hammond CM, White D, Tomic J, Shi Y, Spaner DE. Extracellular calcium sensing promotes human B-cell activation and function. Blood. 2007; 110(12):3985-95. (Methodology) Curr Protoc Cytom. 2001; Chapter 9:Unit 9.8. (Methodology) June CH, Abe R, Rabinovitch PS. Measurement of intracellular calcium ions by flow cytometry. June CH, Moore JS. Measurement of intracellular ions by flow cytometry. Curr Protoc Immunol. 2004; Chapter 5:Unit 5.5. (Methodology) Orbán C, Bajnok A, Vásárhelyi B, Tulassay T, Toldi G. Different calcium influx characteristics upon Kv1.3 and IKCa1 potassium channel inhibition in T helper subsets. Cytometry A. 2014; 85(7):636-41. (Methodology) Roederer M, Bigos M, Nozaki T, Stovel RT, Parks DR, Herzenberg LA. Heterogeneous calcium flux in peripheral T cell subsets revealed by five-color flow cytometry using log-ratio circuitry. Cytometry. 1995; 21(2):187-96. (Methodology) Page 2 of 2 565879 Rev. 1

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