细胞二价铁离子检测探针—FerroOrange

铁离子检测丨从实验思路到检测指标 PDF下载

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性状：可溶于乙腈，甲醇和二甲基亚砜。

纯度（HPLC）：92.0%以上

荧光光谱：符合一致性

铁是生物体内最丰富的过渡金属元素，它参与各种生理过程活动。 近年来，活细胞中的游离铁受到广泛关注， 游离铁最常以其稳定的氧化还原态存在，即亚铁离子 (Fe²⁺) 和铁离子 (Fe³⁺)。对研究者来说，在研究细胞内的还原环境，金属转运蛋白和Fe²⁺的水溶性时，了解Fe²⁺的行为比了解Fe³⁺的行为更为重要。FerroOrange作为一种新型的荧光探针，可对活细胞内的Fe²⁺进行荧光成像。FerroOrange和Fe²⁺反应后的荧光强度上升不可逆，与Fluo-3（货号：F019）这类可以实时监测钙离子的荧光探针有所不同。

FerroOrange检测胞内Fe²⁺

图为将2 μl的1 mmol/l FerroOrange和2 μl的10 mmol/l的各种金属离子添加到1 ml的50 mmol/l HEPES缓冲液（pH 7.4）中，并在室温下反应1小时后测量的荧光强度。

Ex：543 nm Em：580 nm

FerroOrange与各种细胞内的染色试剂共染

用各种染色试剂染色HeLa细胞，清洗细胞。然后将FerroOrange添加到细胞中，并用荧光显微镜观察。

与ER染色试剂共染

＜检测条件＞

FerroOrange: Ex: 561 nm、Em: 570-620 nm

ER Tracker Green（ER染色试剂）: Ex: 488 nm、Em: 510-555 nm

标尺: 10 μm

与线粒体染色试剂共染

＜检测条件＞

FerroOrange: Ex: 561 nm、Em: 570-620 nm

MitoBright Deep Red（线粒体染色试剂）: Ex: 640 nm、Em: 650-700 nm

标尺: 10 μm

与高尔基体染色试剂的共染

＜检测条件＞

FerroOrange: Ex: 561 nm、Em: 570-620 nm

BODIPY FL（高尔基体染色试剂）: Ex: 488 nm、Em: 510-555 nm

标尺: 10 μm

1.细胞接种于荧光培养皿中，在37℃，5% CO₂培养箱中孵育过夜。

2.弃去上清液，并用HBSS或无血清培养基洗涤细胞3次。

3.更换含有药物的培养基，在37℃，5% CO₂培养箱中孵育。

* 请根据药物特性优化孵育时间。

4.加入浓度为1 μmol/l的FerroOrange工作液，在37℃，5% CO₂培养箱中孵育。

* 加入FerroOrange染色剂后请立即观察，不要洗涤。

5.在荧光显微镜下观察细胞。

使用HeLa细胞，通过FerroOrange确认细胞内Fe²⁺的变化

与对照组的细胞相比，用硫酸亚铁 (II) 铵处理的HeLa细胞的FerroOrange荧光强度增加。 相反，在用Bpy处理的细胞中，其荧光强度降低。

因此，证明FerroOrange与细胞内Fe²⁺反应。

A: 对照组

B: 铁剂·硫酸亚铁(II) 铵（终浓度100umol/l）处理

C: 铁螯合剂·2,2'-Bipyridyl (Bpy)（终浓度100umol/l）处理

检测条件 Ex/Em = 561 nm/570-620 nm 比例尺：20 μm

通过流式细胞仪进行定量分析

——FerroOrange的荧光强度可能受细胞密度和细胞种类的影响

——培养基的更换和清洗可能使染料从细胞内泄露

——进行流式定量分析时，需优化实验步骤

1. HeLa cells (1 x105 cells/well) in MEM (10% fetal bovine serum, 1% penicillin-streptomycin) were seeded on a 6 well plate and were cultured at 37oC in a 5% CO2 incubator overnight.
2. The cells were washed with serum-free medium (2 mL) three times. Then, serum-free medium (1 mL) was added to the cells.
3. 10 mmol/L Ammonium iron (II) sulfate (10 μL) was added to wells (The final concentration: 100 μmol/L).
4. To mix Ammonium iron(II) sulfate and serum-free medium, the entire medium was pipetted up from wells and then immediately pipetted back one time.
5. The cells were incubated for 20 min in a 37oC incubator equilibrated with 95% air and 5% CO2, and the cells were washed with HBSS (1 mL) three times.
6. After trypsinization (250 µL), stop the reaction with serum medium (1 mL), 1.25 ml of the cell suspension was transferred to a microcentrifuge tube.
7. The cells suspension was centrifuged at 1,500 rpm for 3 minutes.
8. The supernatant was discarded and HBSS (1 mL) was added to the microcentrifuge tube and suspended by pipetting.
9. The cells suspension was centrifuged at 1,500 rpm for 3 minutes and the supernatant was discarded.
10. 1 μmol/L FerroOrange in MEM (serum-free medium) (300 μL) was added to the cells.
11. The cells were incubated for 15 -30 min in a 37oC incubator equilibrated with 95% air and 5% CO2.
12. The stained cells were passed through a cell strainer and analyze samples using a flow cytometer.

<注意点>

1.清洗对于结果分析的影响

2.染料体积对于结果分析的影响