CFSE is afluorescent dyethat can penetrate the cell membrane. It can react with the free amine group in thecytoskeletonprotein inside the cell, and finally form a protein complex with fluorescence. After entering the cell, CFSE locates in the cell membrane, cytoplasm and nucleus, and the fluorescence staining is strongest in the nucleus^[1]. CFSE dye can be uniformly inherited by the cells with cell division and proliferation, and its attenuation is proportional to the number of cell divisions. This phenomenon can be detected and analyzed by flow cytometry under the excitationlightof 488nm, and can be used to detect the proliferation of cells^[1].

CFSE 工作液的配制
1.1 制备储存液
用 DMSO 配制 10 mM 的 CFSE。如用 0.18 mL DMSO 溶解 1 mg CFSE。
注：CFSE 储存液建议分装后于 -20 ℃ 或 -80 ℃ 避光保存。
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 5-10 μM 的 CFSE 工作液。
注：请根据实际情况调整 CFSE 工作液浓度，且现用现配。

细胞染色
2.1 悬浮细胞：离心收集细胞，加入 PBS 洗涤两次，每次 5 分钟。
贴壁细胞：弃去培养基，加入胰蛋白酶消化细胞。离心弃去上清后，加入 PBS 洗涤两次，每次 5 分钟。
2.2 加入 1 mL CFSE 工作液，室温孵育 15 分钟。
2.3 400 g，4℃ 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，在荧光显微镜或流式细胞仪下检测。

557.46

Solid

C₅₈H₃₈N₂O₂₂

150347-59-4

5(6)-羧基二乙酸荧光素 N-琥珀酰亚胺酯

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)

DMSO : 50 mg/mL(89.69 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 (sealed storage, away from moisture and 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 (3.73 mM); Clear solution

  此方案可获得 ≥ 2.08 mg/mL (3.73 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 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%corn oil

  Solubility: ≥ 2.08 mg/mL (3.73 mM); Clear solution

  此方案可获得 ≥ 2.08 mg/mL (3.73 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: CFDA-SE is an intracellular fluorescent dye with green fluorescence, it can be used to detect strain colonization in vivo.
  Method: For determine strain colonization in the intestine.
  1. Strains are re-suspended in PBS to obtain a cell density of 10⁸CFU/mL.
  2. CFDA-SE (1 mM; 10 μL; 20 min; 37℃; dark) is added to 1 mL of bacterial suspension.
  3. Centrifuge (13000 g, 10 min, 4℃) and wash mixture for three times using sterile PBS to remove excess CFDA-SE.
  4. Strains are then re-suspended in sterile PBS (10⁸CFU/mL) and 1 mL of bacterial suspension is gavaged to SD rats. Day 1 and 3 after gavage, the fluorescence imaging is performed on anesthetized rats who are then sacrificed and different intestinal sections are taken for imaging.
  5. The fluorescence imaging is performed using an IVIS Lumina III Smart Imaging System (PerkinElmer, USA).
• 
  Description: CFDA-SE is an intracellular fluorescent dye with green fluorescence, it can be used to stain bacteria.
  Method: For bacteria adhesion assay.
  1. Testing cells are inoculated into confocal laser dishes at a density of 1×10⁴cells per well and cultured for 24 h.
  2. The bacteria suspension is resuspended with CFDA-SE, then centrifuged with aseptic PBS.
  3. Cultured cells are infected with CFDA-SE labeled bacteria in the presence of drug solutions in dark for 2 h.
  4. Finally, the dishes are placed under confocal laser scanning microscopy (FV3000, Japan) for inspection.
• 
  Description: CFDA-SE is an intracellular fluorescent dye with green fluorescence, it can be used to assess cell binding.
  Method: For monocyte adhesion assay.
  1. Cells are labeled with CFDA-SE (5 μM; 4 h; 37℃) with the HUVECs in a moist atmosphere containing 5% CO₂.
  2. Unbound cells in the dishes are removed by washing 5 times with serum-free medium.
  3. Adherent cells are visualized under an inverted fluorescence microscope (Olympus IX71, Japan).
• 
  Description: CFDA-SE is an intracellular fluorescent dye with green fluorescence, it can be used to detect cell membrane fusion.
  Method: For monocyte adhesion assay.
  1. Testing cells are stained with two different dyes (including CFDA-SE), separately.
  2. Fusion is induced by adding drops of pre-warmed 50% PEG 2000 at 37℃. PEG fusion is terminated by adding pre-warmed DMEM medium containing 15% serum to the tube after one minute. After being centrifuged at 1000 g for 5 min, testing fused cells are obtained.
  3. Use a fluorescence microscope for image.
• 
  Description: CFDA-SE is an intracellular fluorescent dye with green fluorescence, it can be used to determine cell lethality.
  Method: For cell staining (Flow cytometric detection of γδT cell killing rate).
  1. CFDA-SE (10 mg) is precisely added to 1 mL of DMSO solution to prepare a solution with a concentration of 17.9385 mM and diluted according to actual needs.
  2. Culture cells with CFDA-SE at a ratio of 10:1.
  3. Wash cells with PBS and use a microscope for image.