1. Rhod-2 AM 工作液的配制

1.1储备液的配制

将 Rhod-2 AM 溶解于 DMSO 中，得到 5 mM 储备液。

1.2 Rhod-2 AM 的配制工作液

用无血清细胞培养基或 PBS 稀释储备液，得到 5-10 μM 的工作液。

注意：Rhod-2 AM 工作液的浓度请根据实际情况调整

2.细胞染色 (96 孔板)

2.1悬浮细胞

a.1000g 4℃ 离心 3-5 分钟，弃上清。PBS 洗 2 次，每次 5 分钟。细胞密度为 1×106/mL。

b.加入 1 mL 工作液，室温孵育 5-30 分钟。

c.400 g 4℃ 离心 3-4 分钟，弃上清。

d.PBS 洗涤 2 次，每次 5 分钟。

e.重悬细胞用无血清细胞培养基或 PBS。荧光显微镜或流式细胞仪观察。

2.2 贴壁细胞

a.在无菌盖玻片上培养贴壁细胞。

b.从培养基中取出盖玻片并吸出多余的培养基。

c.加入 100 μL 工作液，轻轻摇匀使细胞完全覆盖，然后室温孵育 5-30 分钟。

d.用培养基洗涤 2 次，每次 5 分钟。荧光显微镜观察或流式细胞术。

• 
  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°C 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°C.
  2. After three quick washes with the KSOM medium, the oocytes are imaged using a Nikon fluorescence microscope (Nikon, Tokyo, Japan).

储存条件:    2-8°C       

运输方式:    冰袋运输，根据产品的不同，可能会有相应调整。