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  3. Fluorescent Dye
  4. DALGreen

DALGreen 是一种可穿透细胞、pH 敏感的疏水性自噬检测试剂，能够选择性标记自溶酶体。DALGreen 能够穿透活细胞的质膜并进入自噬体内部。DALGreen 通过 PeT 机制产生荧光，在中性 pH 下荧光被淬灭，在酸性、疏水环境中荧光恢复/增强。DALGreen 的荧光强度可反映自噬活性，会受自噬调节剂影响。DALGreen 具有带有末端氨基的两亲结构，在浓度高达 1.0 μM 时无细胞毒性 (Ex/Em = 405/525 nm)。

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DALGreen

DALGreen Chemical Structure

CAS No. : 2222291-02-1

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DALGreen 相关抗体

Top Publications Citing Use of Products

  • 生物活性

  • 纯度 & 产品资料

  • 参考文献

生物活性

DALGreen is a cell-permeable, pH-sensitive hydrophobic autophagy detection reagent that selectively labels autolysosomes. DALGreen can penetrate the plasma membrane of live cells and enter the interior of autophagosomes. DALGreen generates fluorescence via the PeT mechanism; its fluorescence is quenched under neutral pH conditions, while it is restored/enhanced in acidic, hydrophobic environments. The fluorescence intensity of DALGreen, which reflects autophagic activity, is affected by autophagy modulators. DALGreen has an amphipathic structure with a terminal amino group and exhibits no cytotoxicity at concentrations up to 1.0 μM (Ex/Em = 405/525 nm).

体外研究
(In Vitro)

指南 (以下是我们推荐的方案，本方案仅提供指南，应根据您的具体需要进行修改)。
1. 染料制备
1.1 制备储存液
用无水 DMSO 配制 1 mM 的储备液。
注：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 Hanks' HEPES 缓冲液稀释储存液，配制成 0.1-1 μM 的工作液。
注：请根据实际情况调整工作液浓度，且现用现配。
2. 细胞染色
2.1 移除细胞培养基，用无血清培养基洗涤细胞两次，每次 5-10 分钟。
2.2 加入 0.1-1 μM 的工作液，37°C，孵育 30 分钟。
2.3 弃去上清，用无血清培养基洗涤细胞两次，每次 5-10 分钟。
2.4 将细胞置于无血清培养基中饥饿培养 2 小时至过夜，以诱导自噬。并设置适当的对照组。
2.5. 使用荧光显微镜或流式细胞仪观察荧光信号。


DALGreen (1 μM; 30 min preincubation, 3 h starvation) 可选择性标记 HeLa 细胞中酸化的自溶酶体[1]。
DALGreen (1 μM; 30 min preincubation, 0-6 h post-autophagy induction, 4 h rapamycin treatment) 可标记营养缺乏或经 Rapamycin 处理的 HeLa 细胞中的自噬溶酶体，其荧光强度会在营养缺乏的 6 h 内逐渐升高，以此反映自噬活性[2]。
DALGreen (0.1-1.0 μM; 30 min, followed by 24 h culture without dye) 在浓度高达 1.0 μM 时对 HeLa 细胞无细胞毒性[2]。

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

DALGreen 相关抗体:

Cell Autophagy Assay[1]

Cell Line: HeLa cells
Concentration: 1 μM (DALGreen); 0.1 μM (BafA1)
Incubation Time: 30 min (DALGreen preincubation); 3 h (starvation with/without BafA1)
Result: Showed weak fluorescence in untreated control HeLa cells.
Increased the fluorescent area per cell to a mean of ~6% (n=10) in starved cells.
Almost completely abolished fluorescent signals when treated with BafA1 during starvation, reducing the fluorescent area to near control levels (n=6).

Cell Autophagy Assay[1]

Cell Line: wild-type mouse embryonic fibroblasts (WT MEFs)
Concentration: 1 μM (DALGreen); 0.5 μM (rapamycin)
Incubation Time: 30 min (DALGreen preincubation); 3 h (rapamycin treatment)
Result: Showed no colocalization with WIPI2 puncta (phagophores/early autophagic structures) in rapamycin-treated WT MEFs.
Labeled 61.8% of TagRFP-LC3 puncta (representing late autophagic structures/autolysosomes) in rapamycin-treated cells.

Cell Autophagy Assay[1]

Cell Line: wild-type (WT), Atg5 KO, Atg9 KO, and Ulk1/2 DKO mouse embryonic fibroblasts (MEFs)
Concentration: 1 μM
Incubation Time: 30 min (DALGreen prestain); 5 h (starvation)
Result: Showed a mean fluorescent area of ~70 μm2 (n=9) in starved WT MEFs.
Showed significantly reduced signals in starved Atg5 KO, Atg9 KO, and Ulk1/2 DKO MEFs, with mean fluorescent areas of ~40 μm2 (n=7), ~20 μm2 (n=7), and ~10 μm2 (n=7), respectively.
Showed no significant difference in signals between Atg5 KO, Atg9 KO, and Ulk1/2 DKO MEFs.

Cell Autophagy Assay[1]

Cell Line: wild-type mouse embryonic fibroblasts (WT MEFs)
Concentration: 1 μM (DALGreen); 0.5 μM (rapamycin)
Incubation Time: 30 min (DALGreen prestain); up to 8 h (rapamycin treatment)
Result: Showed a time-dependent increase in fluorescent area per cell after rapamycin treatment.
Reached a mean fluorescent area ratio of ~0.4% by 8 h, significantly higher than the no-treatment control (~0.2%).
Showed an increase in signals that occurred later than the increase in DAPRed signals.

Cell Autophagy Assay[1]

Cell Line: wild-type (WT), Atg5 KO, Atg9 KO, and Ulk1/2 DKO mouse embryonic fibroblasts (MEFs)
Concentration: 1 μM (DALGreen); 10 μM (etoposide)
Incubation Time: 30 min (DALGreen prestain); 10 h (etoposide treatment)
Result: Showed a mean fluorescent area of ~70 μm2 (n=7) in etoposide-treated WT MEFs.
Showed significantly reduced but positive signals in etoposide-treated Atg5 KO and Atg9 KO MEFs (~40 μm2 and ~30 μm2, respectively; n=7 each).
Showed minimal signals (~10 μm2, n=7) in etoposide-treated Ulk1/2 DKO MEFs.
Showed no significant difference in signals between Atg5 KO and Atg9 KO MEFs.

Cell Autophagy Assay[1]

Cell Line: Atg5-deficient mouse embryonic fibroblasts (Atg5 KO MEFs)
Concentration: 1 μM (DALGreen); 10 μM (etoposide)
Incubation Time: 30 min (DALGreen prestain); up to 15 h (etoposide treatment)
Result: Showed a time-dependent increase in fluorescent area per cell after etoposide treatment, with a significant increase starting at 8.5 h.
Reached a mean fluorescent area ratio of ~0.4% by 15 h, significantly higher than the no-treatment control (~0.1%).

Cell Autophagy Assay[2]

Cell Line: HeLa cells
Concentration: 1 μM (30 min preincubation); 1 μM (rapamycin treatment)
Incubation Time: 30 min (preincubation); 0-6 h (post-autophagy induction, nutrient deprivation); 4 h (rapamycin treatment)
Result: Increased over 0 to 6 h of nutrient deprivation, corresponding to increased LC3-II levels and LC3 puncta formation.
Colocalized almost entirely with tagRFP-LC3 in rapamycin-treated cells, indicating staining of acidic autophagic compartments (autolysosomes).

Cell Autophagy Assay[2]

Cell Line: HeLa cells, ULK1/ULK2 double-knockout (DKO) MEF cells
Concentration: 1 μM
Incubation Time: ~5 h (nutrient deprivation with 3-MA treatment); 8 h (rapamycin plus CLQ treatment)
Result: Efficiently quenched by 3-MA treatment in nutrient-deprived HeLa cells.
Showed dramatically reduced staining in ULK1/ULK2 DKO MEFs even with CLQ treatment, compared to wild-type MEFs.

Cell Autophagy Assay[2]

Cell Line: HeLa cells
Concentration: 1 μM
Incubation Time: 5 h (nutrient deprivation with CLQ or bafilomycin A1 treatment)
Result: Enhanced by CLQ treatment.
Completely quenched by bafilomycin A1 treatment.

Cell Cytotoxicity Assay[2]

Cell Line: HeLa cells
Concentration: 0.1-1.0 μM
Incubation Time: 30 min, followed by 24 h culture without dye
Result: Showed no cytotoxicity at concentrations up to 1.0 μM, as measured by cell viability.
体内研究
(In Vivo)

DALGreen (1 μM; immersion; 30 minutes) 因内源性自发荧光的干扰，无法有效用于斑马鱼幼体的体内自噬检测[1]。

MCE has not independently confirmed the accuracy of these methods. They are for reference only.
分子量

430.97

Formula

C23H31ClN4O2
CAS 号
运输条件

Room temperature in continental US; may vary elsewhere.
储存方式

Please store the product under the recommended conditions in the Certificate of Analysis.
纯度 & 产品资料
参考文献

DALGreen 相关分类

  • 摩尔计算器

  • 稀释计算器

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Concentration (start) × Volume (start) = Concentration (final) × Volume (final)

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Help & FAQs
  • Do most proteins show cross-species activity?

    Species cross-reactivity must be investigated individually for each product. Many human cytokines will produce a nice response in mouse cell lines, and many mouse proteins will show activity on human cells. Other proteins may have a lower specific activity when used in the opposite species.

Keywords:

DALGreen2222291-02-1Fluorescent DyeWT MEFsAtg9 KO MEFsautolysosomescanonical autophagyUlk1/2 DKO MEFsendothelial cellsalternative autophagyHeLa cellsautophagic fluxAtg5 KO MEFsInhibitorinhibitorinhibit

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