Rhod-2 is a high-affinity visiblelightexcitation wavelength CaCa²⁺fluorescent probe, Rhod-2, AM is an acetyl methyl ester derivative of Rhod-2, which has cell membrane permeability and can easily enter cells with simple culture. Once it enters the cell, it is sheared by its lactesterase to produce Rhod-2 without membrane permeability, which remains in the cell to perform the corresponding physiological functions. Maximum excitation/emission wavelength: 549/578 nm^[1].

1.Rhod-2 AM 工作液的配制
1.1 制备储存液
用 无水DMSO 配制 5 mM 的 Rhod-2 AM。
1.2 工作液的配制
用预热好的无血清细胞培养基或 PBS 稀释储存液，配制成 1-10 μM 的 Rhod-2 AM 工作液。
注：请根据实际情况调整 Rhod-2 AM 工作液浓度，且现用现配。
2. 细胞染色(悬浮细胞)
2.1 悬浮细胞：在 4℃ 下 1000 g 离心 3-5 分钟，然后丢弃上清液。加入 PBS 洗涤两次，每次 5 分钟。细胞密度在 1×10⁶/mL。
2.2 加入 1 mL Rhod-2 AM 工作液，室温孵育 5-30 分钟。
2.3 400 g，离心 3-4 分钟，弃去上清。
2.4 加入 PBS 洗涤细胞两次，每次 5 分钟。
2.5 用 1 mL 无血清培养基或 PBS 重悬细胞后，使用荧光显微镜或流式细胞仪进行观察。
3. 细胞染色(贴壁细胞)
3.1 将贴壁细胞培养于无菌盖玻片上。
3.2 从培养基中移出盖玻片，吸除多余培养基。
3.3 加入 100 μL 染料工作液，轻轻晃动使其完全覆盖细胞，室温孵育 5-30 分钟。
3.4 吸去染料工作液，用培养基洗 2-3 次，每次 5 分钟 ，使用荧光显微镜或流式细胞仪进行观察。

1123.94

Oil

C₅₂H₅₉BrN₄O₁₉

145037-81-6

Room temperature in continental US; may vary elsewhere.

-20°C, sealed storage, away from moisture and light

*In solvent : -80°C, 6 months; -20°C, 1 month (sealed storage, away from moisture and light)

• 
  Description: Rhod-2 AM is a mitochondrial probe, it can be used to detect mitochondrial Ca²⁺levels with red fluorescence.
  Method: For mitochondrial potential assessment.
  1. After treatment, cells are incubated at 37℃ in dark with 4 μM Rhod-2 AM for 20 min.
  2. Wash cells for three times and re-suspended in Hank’s Balanced Salt Solution without Ca²⁺and Mg²⁺.
  3. Treat cells with REE NPs and additionally stained with 20 nM TMRM, then analysed by flow cytometry (FACS) for mitochondrial membrane assessment. FACS analysis is performed using an LSR II flow cytometer (Becton Dickinson, Mountain View, CA) equipped with a single 488 nm argon laser. Rhod-2 fluorescence are examined using the PE channel; and both forward and side scatters are used to eliminate cellular fragments.
  4. Following treatment, a portion of cells are also fixed with 10% PBS-buffered formaldehyde with 0.1% Triton X-100 for 10 min, then stained with 4’ 6-diamidino-2-phenylindole (DAPI) (blue) and Fluo-3/AM (green) or Rhod-2 (red). Fluorescent images are visualized through confocal laser scanning microscopy.
• 
  Description: Rhod-2 AM is a mitochondrial probe, it can be used to detect mitochondrial Ca²⁺levels with red fluorescence.
  Method: For measurement of mitochondrial Ca²⁺levels.
  1. Cells are incubated with Rhod-2 AM (10 μM; 2 h; dark).
  2. Wash cells twice with PBS, and then resuspended in PBS.
  3. Fluorescence is determined by flow cytometry, and fluorescence photomicrographs are taken using a fluorescence microscope.
• 
  Description: Rhod-2 AM is a Ca²⁺fluorescence probe that specifically aggregates in mitochondria because of its positive charge, it can be used to track mitochondrial Ca²⁺uptake in cells.
  Method: For measurement of mitochondrial Ca²⁺uptake in oocytes.
  1. Around 40 oocytes are incubated with Rhod-2 AM (6 μM) in the KSOM medium for 30 min at 37℃.
  2. After three quick washes with the KSOM medium, the oocytes are imaged using a Nikon fluorescence microscope (Nikon, Tokyo, Japan).