H2DCFDA (DCFH-DA) is a cell-permeable probe used to detect intracellularreactive oxygen species(ROS)(Ex/Em=488/525 nm)^[1].

Guidelines (Following is our recommended protocol. This protocol only provides a guideline, and should be modified according to your specific needs).
1. H2DCFDA is dissolved in DMSO to obtain a 10 mM stock solutions and further diluted before use.
2. Cells are incubated with 5 μM H2DCFDA solution in PBS in the dark for 30 min at 37℃, then harvested with 0.05% trypsin-EDTA solution, suspended in a fresh medium, and immediately analyzed with flow cytometer.
3. Along with the H2DCFDA probe, if indicated, use ROS-insensitive modification of the fluorescein dye DCFDA as a positive control. The staining procedure is the same as for the H2DCFDA^[1].

487.29

Solid

C₂₄H₁₆Cl₂O₇

4091-99-0

Room temperature in continental US; may vary elsewhere.

-20°C, protect from light

*In solvent : -80°C, 6 months; -20°C, 1 month (protect from light)

DMSO : 125 mg/mL(256.52 mM;Need ultrasonic)

Ethanol : 20 mg/mL(41.04 mM;Need ultrasonic)

请根据产品在不同溶剂中的溶解度选择合适的溶剂配制储备液；一旦配成溶液，请分装保存，避免反复冻融造成的产品失效。
储备液的保存方式和期限：-80℃, 6 months; -20℃, 1 month (protect from light)。-80℃ 储存时，请在 6 个月内使用，-20℃ 储存时，请在 1 个月内使用。

请根据您的实验动物和给药方式选择适当的溶解方案。以下溶解方案都请先按照In Vitro方式配制澄清的储备液，再依次添加助溶剂：

——为保证实验结果的可靠性，澄清的储备液可以根据储存条件，适当保存；体内实验的工作液，建议您现用现配，当天使用； 以下溶剂前显示的百
分比是指该溶剂在您配制终溶液中的体积占比；如在配制过程中出现沉淀、析出现象，可以通过加热和/或超声的方式助溶

• 1.

  请依序添加每种溶剂： 10% DMSO    40%PEG300   5%Tween-80   45% saline

  Solubility: 2.08 mg/mL (4.27 mM); Suspended solution; Need ultrasonic

  此方案可获得 2.08 mg/mL (4.27 mM) 的均匀悬浊液，悬浊液可用于口服和腹腔注射。

  以 1 mL 工作液为例，取 100 μL 20.8 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀；向上述体系中加入50 μL Tween-80，混合均匀；然后继续加入 450 μL生理盐水定容至 1 mL。

  将 0.9 g 氯化钠，完全溶解于 100 mL ddH2O 中，得到澄清透明的生理盐水溶液
• 2.

  请依序添加每种溶剂： 10% DMSO    90% (20%SBE-β-CDin saline)

  Solubility: ≥ 2.08 mg/mL (4.27 mM); Clear solution

  此方案可获得 ≥ 2.08 mg/mL (4.27 mM，饱和度未知) 的澄清溶液。

  以 1 mL 工作液为例，取 100 μL 20.8 mg/mL 的澄清 DMSO 储备液加到 900 μL20% 的 SBE-β-CD 生理盐水水溶液中，混合均匀。

  将 2 g 磺丁基醚 β-环糊精加入 5 mL 生理盐水中，再用生理盐水定容至 10 mL，完全溶解，澄清透明
• 3.

  请依序添加每种溶剂： 10% DMSO    90%corn oil

  Solubility: ≥ 2.08 mg/mL (4.27 mM); Clear solution

  此方案可获得 ≥ 2.08 mg/mL (4.27 mM，饱和度未知) 的澄清溶液，此方案不适用于实验周期在半个月以上的实验。

  以 1 mL 工作液为例，取 100 μL 20.8 mg/mL 的澄清 DMSO 储备液加到 900 μL玉米油中，混合均匀。

• 
  Description: H2DCFDA (DCFH-DA) is a ROS production probe with green fluorescence.
  Method: For cell staining.
  1. Stain cells with H2DCFDA.
  2. Capture staining cells by spinning-disk confocal microscopy (every 5 min for 6 h).
• 
  Description: H2DCFDA (DCFH-DA) is a ROS production probe with green fluorescence.
  Method: For cell staining.
  1. Remove the cell culture medium and replaced by fresh medium containing H2DCFDA and incubated with cultured cells for 30 min.
  2. Wash cells with PBS for three times.
  3. Use a confocal laser scanning microscopy (FV1000, Olympus Company, Japan) to detect the intracellular ROS generation.
• 
  Description: H2DCFDA (DCFH-DA) is a ROS production probe with green fluorescence.
  Method: For cell staining.
  1. Testing cells are seeded into a 6-well microplate at a density of 10⁵/well and cultured overnight at 37℃ in 5% CO₂. Fresh medium with 50 μg/mL of different materials is added to each well and cultured for 16 h.
  2. Wash cells with PBS and stain cells with H2DCFDA (20 μM; 1 h; 37℃; dark).
  3. Cells are trypsinized, washed, resuspended in PBS (0.2 mL), analyzed by a flow cytometer and use a confocal laser scanning microscopy (FV1000, Olympus, Tokyo, Japan) for image.
• 
  Description: H2DCFDA (DCFH-DA) is a ROS production probe with green fluorescence.
  Method: For cell staining.
  1. Wash cultured cells with PBS for three times.
  2. Treat testing cells with H2DCFDA in culture medium for 0.5 h in a cell incubator.
  3. Use a confocal laser scanning microscopy (Olympus, FV3000) for image. Cells are analyzed on a BD LSRI Fortessa flow cytometer analyzer to quantify results.
• 
  Description: H2DCFDA (DCFH-DA) is a ROS production probe with green fluorescence.
  Method: For cell staining.