生物活性
钙离子荧光探针Rhod-2, AM是一种用于钙通量测定的试剂（λex=552 nm，λem=581 nm）。钙测量对于许多生物学研究至关重要。结合 Ca2+ 时显示光谱响应的荧光探针使研究人员能够通过使用荧光显微镜、流式细胞术、荧光光谱和荧光酶标仪来研究细胞内游离 Ca2+ 浓度的变化。
PREPARATION OF STOCK SOLUTIONS
Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles.
Rhod-2 AM Stock Solution
Prepare a 2 to 5 mM stock solution of Rhod-2 AM in high-quality, anhydrous DMSO.
PREPARATION OF WORKING SOLUTION
Rhod-2 AM Working Solution
On the day of the experiment, either dissolve Rhod-2 AM in DMSO or thaw an aliquot of the indicator stock solution to room temperature. Prepare a dye working solution of 2 to 20 µM in a buffer of your choice (e.g., Hanks and Hepes buffer) with 0.04% Pluronic® F-127. For most cell lines, Rhod-2 AM at a final concentration of 4-5 μM is recommended. The exact concentration of indicators required for cell loading must be determined empirically.
Note The nonionic detergent Pluronic® F-127 is sometimes used to increase the aqueous solubility of Rhod-2 AM.
Note If your cells contain organic anion-transporters, 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.
SAMPLE EXPERIMENTAL PROTOCOL
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 cells in growth medium overnight.
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On the next day, add 1X Rhod-2 AM working solution into your cell plate.
Note If your compound(s) interfere with the serum, replace the growth medium with fresh HHBS buffer before dye-loading.
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Incubate the dye-loaded plate in a cell incubator at 37 °C for 30 to 60 minutes.
Note Incubating the dye for longer than 1 hour can improve signal intensities in certain cell lines.
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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 any excess probes.
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Add the stimulant as desired and simultaneously measure fluorescence using either a fluorescence microscope equipped with a TRITC filter set or a fluorescence plate reader containing a programmable liquid handling system such as an FDSS, FLIPR, or FlexStation, at Ex/Em = 540/590 nm cutoff 570 nm.
化学性质
| 分子量 |
1123.96 |
| 分子式 |
C52H59BrN4O |
| CAS号 |
145037-81-6
|
| 溶解性（25°C） |
DMSO 1~5mM |
| 储存条件 |
-20°C, protect from light, dry, sealed
|
| 运输方式 |
冰袋运输，根据产品的不同，可能会有相应调整。 |
储备液配制
*下述溶液配置方法仅为基于分子量计算出的理论值。不同产品在配置溶液前，需考虑其在不同溶剂中的溶解度限制。
| Concentration / Solvent Volume / Mass |
1 mg |
5 mg |
10 mg |
| 1 mM |
0.8897 mL |
4.4486 mL |
8.8971 mL |
| 5 mM |
0.1779 mL |
0.8897 mL |
1.7794 mL |
| 10 mM |
0.089 mL |
0.4449 mL |
0.8897 mL |
不同实验动物依据体表面积的等效剂量转换表（参考来源于公开文献）
|
小鼠 |
大鼠 |
兔 |
豚鼠 |
仓鼠 |
狗 |
| 重量 (kg) |
0.02 |
0.15 |
1.8 |
0.4 |
0.08 |
10 |
| 体表面积 (m2) |
0.007 |
0.025 |
0.15 |
0.05 |
0.02 |
0.5 |
| Km 系数 |
3 |
6 |
12 |
8 |
5 |
20 |
| 动物 A (mg/kg) = 动物 B (mg/kg) × |
动物 B的Km系数
|
| 动物 A的Km系数 |
例如，依据体表面积折算法，将化合物用于小鼠的剂量20 mg/kg 换算成大鼠的剂量，需要将20 mg/kg 乘以小鼠的Km系数（3），再除以大鼠的Km系数（6），得到化合物用于大鼠的等效剂量为10 mg/kg。
参考文献
[1] Xudong Peng, et al. BMC Ophthalmol. Phospholipase Cγ2 is critical for Ca 2+ flux and cytokine production in anti-fungal innate immunity of human corneal epithelial cells
[2] Cynthia Brisac, et al. J Virol. Calcium flux between the endoplasmic reticulum and mitochondrion contributes to poliovirus-induced apoptosis
[3] Xiaoyu D, et al. Plasma Science and Technology. Measurement of cytoplasmic Ca2+ concentration in Saccharomyces cerevisiae induced by air cold plasma
蛋白质研究