DRAQ5；DRAQ7；Anthraquinone dye 蒽醌染料；DNA染料；7-AAD；细胞周期分析；染色体倍性分析；有核/无核细胞区分；

产品信息

产品描述

DRAQ5是一种细胞通透性的远红外DNA染料，能联合其它常见的标记染料比如：GFP或FITC，用于固定或非固定/活细胞的染色。与任何其它细胞通透性的DNA嵌入染料一样，DRAQ5在长期实验中可能抑制细胞分裂，需测定染料可能的任何效应。

DRAQ5能用于流式细胞仪、活细胞成像和基于细胞的各种实验，DRAQ5高度兼容于许多仪器平台上的标准实验方案。DRAQ5染色包含这些优势：1）以便捷的即用型水溶性溶液形式提供；2）能被活细胞快速摄取，提供高水平的细胞核辨别力；3）无光漂白效应；4）适用于绝大多数细胞，真核和原核来源：哺乳动物、细菌、寄生虫、植物等；5） 流式细胞检测中与常见的FITC/GFP+PE结合实验无需做荧光补偿；6）不需要RNase处理；7）与DNA结合后没有荧光增强，因此，测量的荧光与细胞内的核DNA量呈比例和化学计量比。测量不到mtDNA结合，与RNA结合可忽略。8）兼容于台式流式细胞仪、激光扫描细胞仪和非紫外激光扫描和基于灯泡的共聚焦显微镜。

染料特性

1）   化学名称：1, 5-bis{[2-(di-methylamino)ethyl]amino}-4, 8-dihydroxyanthracene-9, 10-dione 1,5-双{[2-(二甲基氨基)乙基]氨基}-4, 8-二羟基蒽-9,10-二酮

2）   分子量：412.54

3）   激发波长：最理想647nm处（Eλmax= 646 nm）； 次理想488，514，568和633nm处

4）   发射波长（取决于仪器）：665nm至红外（Emλmax= 681nm/697nm，掺入 dsDNA）

          与可见光区比如：GFP和FITC，最小重叠；发射滤片可能包括：695L, 715LP 或780 LP

保存与运输方法

保存：2-8°C避光保存，2年有效。不可冻存！

运输：室温运输。

注意事项

1）DRAQ5（5mM）不可冻存，但冻存会导致染料析出，且很难（不可能）重溶回溶液。

2）DRAQ5是Biostatus Limited的商标名。

3）荧光染料都存在淬灭的问题，保存和操作过程中注意避光。

4）为了您的安全和健康，请穿实验服并戴一次性手套操作。

染色步骤

1）根据所需步骤执行表面染色；

2）PBS清洗细胞两次。叠氮钠影响DRAQ5染色，建议用PBS（不含钙镁或叠氮钠）或细胞培养基来染色。

3）稀释DRAQ5到所需浓度。推荐做浓度滴定来确定最佳的工作浓度。用于流式细胞仪和显微成像检测， 1:200~1:1000的稀释倍数（5-25µM）能获得良好的染色结果。

4）室温避光孵育10-15min。

5）用装有633红色激发器的细胞仪进行细胞分析。做细胞周期分析时，用带680LP或715LP滤片的Alexa 700通道可能有助于分辨发射峰。

延伸阅读（比较PI和DRAQ5的细胞周期分析）

Yuan C. M. et al. DRAQ5-Based DNA content analysis of hematolymphoid cell subpopulations discriminated by surface antigens and light scatter properties. Cytometry B 58, 47–52 (2004).

DNA Staining

For DRAQ5 staining, 3 × 105 cells in 250 μL of PBS, previously stained for surface antigens were incubated with 2 μL of DRAQ5 for 5 min at room temperature and protected from bright light.

For PI staining, reagents from CycleTEST Plus (BD), were used according to manufacturer’s recommendations. In summary, cells were exposed to a trypsin solution for 10 min at room temperature, followed by a solution containing trypsin and RNAse A, and subsequently incubated in PI for 10 min at 4°C at a final concentration of 125 μg/ml.

Data Acquisition

Cells were acquired on a FACSCalibur four-color flow cytometer (BD) equipped with both a 488-nm argon laser and a 635-nm diode laser. The data were acquired using CellQuest software (BD). Acquisition of the whole sample was performed without gating to exclude cell doublets and debris. The total number of cells collected in each case ranged from 40 × 103 to 50 × 103.

Data Analysis

For DRAQ5 and PI comparisons, DNA cell cycle analysis was performed on unselected populations. DRAQ5-based DNA content analysis of discrete marrow subpopulations was performed by identifying and selecting those subpopulations by CD45 expression and side scatter properties. The following cell types were identified: lymphocytes, monocytes, mature granulocytes, immature granulocytes, nucleated erythroid cells, and early precursors (blast region). The latter population was defined by CD34 expression, along with intensity of CD45 expression and side scatter. Each subpopulation’s data were saved as separate Flow Cytometry Standard files using CellQuest software (BD), and analyzed for cell cycle phases using Modfit LT 3.1 (Verity Software House, Topsham, MA).

DNA content analysis included determination of the mean channel fluorescence and the coefficient of variation (CV) of the G1 peaks, number of cell doublets, and S-phase fractions. All data were generated using the Autoanalysis function of the Modfit LT 3.1 program, with active aggregates and debris modeling, according to software manufacturer’s recommendations. Paired t-test was used for statistical calculations.

For the purposes of data display and creation of figures, dot plots were generated using WinMDI 2.8 (Joe Trotter, Scripps Institute).

Table 1. Comparison Between DRAQ5 and PI Derived Cell Cycle Analysis Parameters in a Variety of Hematolymphoid Samples

Figure 1

Examples of two cases stained in duplicate with DRAQ5 and PI. The first case exhibits a single G0/G1 peak (A) and the second case exhibits two G0/G1 peaks (B), representing aneuploidy. CV1 corresponds to the first G0/G1 peak located approximately at channel 50 and CV2 corresponds to the second G0/G1 peak (aneuploid G0/G1 peak). DRAQ5 produces slightly larger CVs than PI in both cases (A,B). An identical DNA index (DI) of the second G0/G1 peak (B) was obtained with both DRAQ5 and PI staining.

常见问题

1）DRAQ5能用于哪种细胞类型的染色？

2）DRAQ5用于哪些类型的实验？

3）如果检测样本内含更多细胞，需要加入更多的DRAQ5？

4）DRAQ5能染色线粒体或RNA？

5）为什么DRAQ5染色的细胞不需要清洗？

6）DRAQ5染色可用抗荧光封片剂？

答：是的。

7）如果DRAQ5用于活细胞染色，可以做长期实验吗？

答：不行。

8）哪些荧光素能与DRAQ5同时使用？

9）DRAQ5工作液能保存多久？

答：建议稀释后的DRAQ5工作液尽快使用，不要保存工作液。

相关产品

DRAQ5；DRAQ7；Anthraquinone dye 蒽醌染料；DNA染料；7-AAD；细胞周期分析；染色体倍性分析；有核/无核细胞区分；

产品信息

产品描述

DRAQ5是一种细胞通透性的远红外DNA染料，能联合其它常见的标记染料比如：GFP或FITC，用于固定或非固定/活细胞的染色。与任何其它细胞通透性的DNA嵌入染料一样，DRAQ5在长期实验中可能抑制细胞分裂，需测定染料可能的任何效应。

DRAQ5能用于流式细胞仪、活细胞成像和基于细胞的各种实验，DRAQ5高度兼容于许多仪器平台上的标准实验方案。DRAQ5染色包含这些优势：1）以便捷的即用型水溶性溶液形式提供；2）能被活细胞快速摄取，提供高水平的细胞核辨别力；3）无光漂白效应；4）适用于绝大多数细胞，真核和原核来源：哺乳动物、细菌、寄生虫、植物等；5） 流式细胞检测中与常见的FITC/GFP+PE结合实验无需做荧光补偿；6）不需要RNase处理；7）与DNA结合后没有荧光增强，因此，测量的荧光与细胞内的核DNA量呈比例和化学计量比。测量不到mtDNA结合，与RNA结合可忽略。8）兼容于台式流式细胞仪、激光扫描细胞仪和非紫外激光扫描和基于灯泡的共聚焦显微镜。

染料特性

1）   化学名称：1, 5-bis{[2-(di-methylamino)ethyl]amino}-4, 8-dihydroxyanthracene-9, 10-dione 1,5-双{[2-(二甲基氨基)乙基]氨基}-4, 8-二羟基蒽-9,10-二酮

2）   分子量：412.54

3）   激发波长：最理想647nm处（Eλmax= 646 nm）； 次理想488，514，568和633nm处

4）   发射波长（取决于仪器）：665nm至红外（Emλmax= 681nm/697nm，掺入 dsDNA）

          与可见光区比如：GFP和FITC，最小重叠；发射滤片可能包括：695L, 715LP 或780 LP

保存与运输方法

保存：2-8°C避光保存，2年有效。不可冻存！

运输：室温运输。

注意事项

1）DRAQ5（5mM）不可冻存，但冻存会导致染料析出，且很难（不可能）重溶回溶液。

2）DRAQ5是Biostatus Limited的商标名。

3）荧光染料都存在淬灭的问题，保存和操作过程中注意避光。

4）为了您的安全和健康，请穿实验服并戴一次性手套操作。

染色步骤

1）根据所需步骤执行表面染色；

2）PBS清洗细胞两次。叠氮钠影响DRAQ5染色，建议用PBS（不含钙镁或叠氮钠）或细胞培养基来染色。

3）稀释DRAQ5到所需浓度。推荐做浓度滴定来确定最佳的工作浓度。用于流式细胞仪和显微成像检测， 1:200~1:1000的稀释倍数（5-25µM）能获得良好的染色结果。

4）室温避光孵育10-15min。

5）用装有633红色激发器的细胞仪进行细胞分析。做细胞周期分析时，用带680LP或715LP滤片的Alexa 700通道可能有助于分辨发射峰。

延伸阅读（比较PI和DRAQ5的细胞周期分析）

Yuan C. M. et al. DRAQ5-Based DNA content analysis of hematolymphoid cell subpopulations discriminated by surface antigens and light scatter properties. Cytometry B 58, 47–52 (2004).

DNA Staining

For DRAQ5 staining, 3 × 105 cells in 250 μL of PBS, previously stained for surface antigens were incubated with 2 μL of DRAQ5 for 5 min at room temperature and protected from bright light.

For PI staining, reagents from CycleTEST Plus (BD), were used according to manufacturer’s recommendations. In summary, cells were exposed to a trypsin solution for 10 min at room temperature, followed by a solution containing trypsin and RNAse A, and subsequently incubated in PI for 10 min at 4°C at a final concentration of 125 μg/ml.

Data Acquisition

Cells were acquired on a FACSCalibur four-color flow cytometer (BD) equipped with both a 488-nm argon laser and a 635-nm diode laser. The data were acquired using CellQuest software (BD). Acquisition of the whole sample was performed without gating to exclude cell doublets and debris. The total number of cells collected in each case ranged from 40 × 103 to 50 × 103.

Data Analysis

For DRAQ5 and PI comparisons, DNA cell cycle analysis was performed on unselected populations. DRAQ5-based DNA content analysis of discrete marrow subpopulations was performed by identifying and selecting those subpopulations by CD45 expression and side scatter properties. The following cell types were identified: lymphocytes, monocytes, mature granulocytes, immature granulocytes, nucleated erythroid cells, and early precursors (blast region). The latter population was defined by CD34 expression, along with intensity of CD45 expression and side scatter. Each subpopulation’s data were saved as separate Flow Cytometry Standard files using CellQuest software (BD), and analyzed for cell cycle phases using Modfit LT 3.1 (Verity Software House, Topsham, MA).

DNA content analysis included determination of the mean channel fluorescence and the coefficient of variation (CV) of the G1 peaks, number of cell doublets, and S-phase fractions. All data were generated using the Autoanalysis function of the Modfit LT 3.1 program, with active aggregates and debris modeling, according to software manufacturer’s recommendations. Paired t-test was used for statistical calculations.

For the purposes of data display and creation of figures, dot plots were generated using WinMDI 2.8 (Joe Trotter, Scripps Institute).

Table 1. Comparison Between DRAQ5 and PI Derived Cell Cycle Analysis Parameters in a Variety of Hematolymphoid Samples

Figure 1

Examples of two cases stained in duplicate with DRAQ5 and PI. The first case exhibits a single G0/G1 peak (A) and the second case exhibits two G0/G1 peaks (B), representing aneuploidy. CV1 corresponds to the first G0/G1 peak located approximately at channel 50 and CV2 corresponds to the second G0/G1 peak (aneuploid G0/G1 peak). DRAQ5 produces slightly larger CVs than PI in both cases (A,B). An identical DNA index (DI) of the second G0/G1 peak (B) was obtained with both DRAQ5 and PI staining.

常见问题

1）DRAQ5能用于哪种细胞类型的染色？

2）DRAQ5用于哪些类型的实验？

3）如果检测样本内含更多细胞，需要加入更多的DRAQ5？

4）DRAQ5能染色线粒体或RNA？

5）为什么DRAQ5染色的细胞不需要清洗？

6）DRAQ5染色可用抗荧光封片剂？

答：是的。

7）如果DRAQ5用于活细胞染色，可以做长期实验吗？

答：不行。

8）哪些荧光素能与DRAQ5同时使用？

9）DRAQ5工作液能保存多久？

答：建议稀释后的DRAQ5工作液尽快使用，不要保存工作液。

相关产品