在所有的钙离子指示剂中，Rhod 2荧光信号的波长最长。它具有和罗丹明类似的激发和发射波长，分别为557 nm和581 nm。这样的激发波长使得我们很容易就能找到氩和氪光源的激光。尽管有人认为Rhod 2的荧光信号仅仅将钙复合物的荧光增加了数倍，但是AAT Bioquest的Rhod 2由于具有很高的纯度，能有效地将钙荧光信号增加80-100倍左右，使得它的信号强度在所有的钙离子探针里面是最强的。所以，我们强烈建议使用Rhod 2作为探针用激光显微镜来检测细胞内的钙离子情况。有报道称，特别是在神经组织切片培养方面，Rhod 2具有定位点的轮廓线更加清晰的特点。Rhod 2和钙离子的解离常数为（Kd＝1.0 mM），在所有的钙离子荧光探针中是最高的，为监测钙离子浓度提供了一个广阔的范围。Rhod 2-AM是一种Rhod 2的乙酰甲酯衍生物，能非常容易的通过AM法负载到细胞内。
- 1. Load Cells with Calcium Indicator AM Esters:
AM esters are the non-polar esters that 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 cell non-invasively. However, cautions must be excised when AM esters are used since they are susceptible to hydrolysis, particularly in solution. They should be reconstituted in high-quality, anhydrous dimethylsulfoxide (DMSO). DMSO stock solutions should 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 AM esters into live cells. This protocol only provides a guideline, and should be modified according to your specific needs.
- Prepare a 2 to 5 mM AM esters stock solution in high-quality, anhydrous DMSO.
- On the day of the experiment, either dissolve calcium indicators solid in DMSO or thaw an aliquot of the indicator stock solutions to room temperature. Prepare a working solution of 2 to 20 µM in the buffer of your choice (such as Hanks and Hepes buffer) with 0.04%Pluronic® F-127. For most cell lines we recommend the final concentration of calcium indicators be 4-5 uM. The exact concentration of indicators required for cell loading must be determined empirically. To avoid any artifacts caused by overloading and potential dye toxicity, it is recommended to use the minimal probe concentration that can yield sufficient signal strength.
Note:The nonionic detergent Pluronic® F-127 is sometimes used to increase the aqueous solubility of calcium indicatorAMesters.
- If your cells (such as CHO cells) containing the organic anion-transports, probenecid (2–5 mM) or sulfinpyrazone (0.2–0.5 mM) may be added to the the dye working solution (final in well concentration will be 1-2.5 mM for probenecid, or 0.1 -0.25 mM for sulfinpyrazone) to reduce the leakage of the de-esterified indicators.
- Add equal volume of the dye working solution (from Step b or c) into your cell plate.
- Incubate the dye-loading plate room at temperature or 37 °C for 20 minutes (especially Fluo-8 AM) to 2 hours, and then incubate the plate at room temperature for another 30 minutes.
Note1: Decreasing the loading temperature might reduce the compartmentalization of the indictor.
Note2: Incubate the Cal-520 AM longer than 2 hours gives better signal intensity for some cell lines.
- Replace the dye working solution with HHBS or buffer of your choice (containing an anion transporter inhibitor, such as 1 mM probenecid, if applicable) to remove excess probes.
- Run the experiments at desired Ex/Em wavelengths (see Table 1).
2. Measure Intracellular Calcium Responses
Figure 1.Response of endogenous P2Y receptor to ATP in CHO-M1 cells withoutprobenecid.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 37oC for 2 hours. ATP (50µL/well) was added by FlexStation (Molecular Devices) to achieve the final indicated concentrations.