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Use of Cal-520® AM, Cal-590™ AM, or Cal-630™ AM Esters

1. Load Cells with Cal-520®, Cal-590™ or Cal-630™ AM Esters:

AM esters are non-polar esters that can readily cross live cell membranes, and rapidly hydrolyzed by cellular esterases inside live cells. AM esters are widely used for loading a variety of polar fluorescent probes into live cells noninvasively. However, cautions must be exercised when AM esters are used since they are susceptible to hydrolysis, particularly in solution. They should be reconstituted just before use in high-quality, anhydrous dimethylsulfoxide (DMSO). DMSO stock solutions may be stored desiccated at –20 °C and protected from light. Under these conditions, AM esters should be stable for several months. Following is our recommended protocol for loading Cal-520® AM,Cal-590™ AM or Cal-630™ AM  esters into live cells. This protocol only provides a guideline, and should be modified according to your specific needs.

a)       Prepare a 2 to 5 mM stock solution of Cal-520® AM, Cal-590™ AM or Cal-630™ AM esters in high-quality, anhydrous DMSO.

b)       On the day of the experiment, either dissolve Cal-520® AM, Cal-590™ AM or Cal-630™ AM in DMSO or thaw an aliquot of the indicator stock solution to room temperature. Prepare a dye working solution of 10 to 20 µM in Hanks and Hepes buffer (HHBS) or the buffer of your choice with 0.04% Pluronic® F-127. The exact concentration of the indicator required for cell loading must be determined empirically.

Note: The nonionic detergent Pluronic® F-127 is sometimes used to increase the aqueous solubility of Cal-520® AM, Cal-590™ AM or Cal-630™ AM esters. A variety of Pluronic® F-127 solutions can be purchased from AAT Bioquest.

c)       If your cells (such as CHO cells) contain organic anion-transports, then probenecid (1-2 mM) may be added to the dye working solution (final in well concentration will be 0.5-1 mM) to reduce leakage of the de-esterified indicators.

Note: A variety of ReadiUse™ probenecid including water soluble sodium salt and stabilized solution can be purchased from AAT Bioquest

d)       Add equal volume of the dye working solution (from Step b or c) into your cell plate.

e)       Incubate the dye-loading plate in a cell incubator for 60 to 90 minutes, and then incubate the plate at room temperature for another 30 minutes.

Note: Incubating the dye longer than 2 hours gives better signal intensity for some cell lines.

f)        Replace the dye working solution with HHBS or if applicable, a buffer of your choice that contains an anion transporter inhibitor, such as 1 mM probenecid, to remove excess probes.

g)       Run the experiments at Ex/Em = 490/525 nm (for Cal-520® AM), 540/590 nm (for Cal-590™ AM) or 600/640 nm (for Cal-630™ AM).

2. Measure Intracellular Calcium Responses:

Figure 1. Response of endogenous P2Y receptor to ATP in CHO-M1 cells without probenecid. CHO-M1 cells were seeded overnight at 40,000 cells per 100 µL per well in a 96-well black wall/clear bottom costar plate. 100 µl of 4 µM Fluo-3 AM, Fluo-4 AM or Cal 520® AM in HHBS were added into the wells, and the cells were incubated at 37 °C for 2 hour. The dye loading medium were replaced with 100 µl HHBS, 50 µl of 300 µM ATP were added, and then imaged with a fluorescence microscope (Olympus IX71) using FITC channel.

Figure 2. ATP-stimulated calcium response of endogenous P2Y receptor in CHO-K1 cells measured with Cal-520® or Fluo-4 AM. CHO-K1cells were seeded overnight in 50,000 cells per 100 µL per well in a 96-well black wall/clear bottom costar plate. 100 µL of 5 µM Fluo-4 AM or the Cal-520® AM with (A) or without (B) 2.5 mM probenecid was added into the cells, and the cells were incubated at 37^oC for 2 hours.  ATP (50µL/well) was added by FlexStation (Molecular Devices) to achieve the final indicated concentrations.

Figure 3. Response of endogenous P2Y receptor to ATP in CHO-K cells. CHO-K cells were seeded overnight at 40,000 cells per 100 µL per well in a 96-well black wall/clear bottom costar plate. 100 µl of 4 µM Cal 590™ AM or Cal 630™ AM in HHBS with 1 mM probenecid were added into the wells, and the cells were incubated at 37 °C for 2 hour. The dye loading mediums were replaced with 100 µl HHBS and 1 mM probenecid , then imaged with a fluorescence microscope (Olympus IX71) using TRITC channel before and after adding 50 µl of 300 µM ATP.

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