YF®640 TUNEL Apoptosis Kit (Far red fluorescence)|US Everbright Inc.

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YF®640 TUNEL Apoptosis Kit (Far red fluorescence)

Product No.:T6063

Product specification:20T/50T

Catalogue price（RMB）:1800/3200

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Product overview
Instructions
Analysis report
MSDS
Asked questions
Citations and references
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Product overview:

Catalog Number and Product Size:

Catalog Number	Product name	Ex/Em (nm)	Size
T6013S	YF^®488 TUNEL Apoptosis Kit (Green fluorescence)	490/515	20T
T6013L	YF^®488 TUNEL Apoptosis Kit (Green fluorescence)	490/515	50T
T6039S	YF^®555 TUNEL Apoptosis Kit (Orange red fluorescence)	555/565	20T
T6039L	YF^®555 TUNEL Apoptosis Kit (Orange red fluorescence)	555/565	50T
T6014S	YF^®594 TUNEL Apoptosis Kit (Red fluorescence)	590/617	20T
T6014L	YF^®594 TUNEL Apoptosis Kit (Red fluorescence)	590/617	50T
T6063S	YF^®640 TUNEL Apoptosis Kit (Far red fluorescence)	642/662	20T
T6063L	YF^®640 TUNEL Apoptosis Kit (Far red fluorescence)	642/662	50T
T6067S	Cy3 TUNEL Apoptosis Kit	555/565	20T
T6067L	Cy3 TUNEL Apoptosis Kit	555/565	50T

Contents:

Size Component	20T	50T
A. TUNEL Equilibration Buffer	2×1 mL	5 mL
B. YF^®488/555/594/640/Cy3 TUNEL Reaction Buffer	1 mL	2×1.25 mL
C. TdT Enzyme	20 μL	50 μL
D. Proteinase K (2 mg/mL)	40 μL	100 μL
E. DNase I (2 U/μL)	5 μL	13 μL
F. 10 × DNase I Buffer	100 μL	260 μL

Storage
Store at -20°C. Component B should be protected from light to avoid repeated freezing and thawing. Expiration date marked on the outer packing.

Description

When cells undergo apoptosis, some DNA endonuclease will be activated, and these endonuclease will cut the genomic DNA between nucleosomes and generate 180 bp-200 bp DNA fragment, which is represented by the specific Ladder pattern presented in agarose gel electrophoresis. Genomic DNA double-strand or single-strand breaks will produce a large number of sticky 3'-OH ends, which can be combined with YF^®/Cy-dUTP under the catalysis of deoxyribonucleotide terminal transferase (TdT), thereby passing through Fluorescence microscopy or flow cytometry can directly detect apoptotic cells. This method is called Terminal-deoxynucleotidyl transferase mediated nick end labeling (TUNEL). Since normal or proliferating cells have little DNA breakage and thus no 3'-OH formation, they are rarely stained. The Tunel method can perform in situ staining of intact single apoptotic cell nuclei or apoptotic bodies, which can accurately reflect the typical biochemical and morphological characteristics of apoptosis, and can detect a very small amount of apoptotic cells. widely used in research.
This kit has a wide range of applications and can be used to detect apoptosis in frozen or paraffin sections, as well as in cultured adherent cells or suspension cells. Selectively detects apoptotic cells rather than necrotic cells or cells with DNA strand breaks caused by irradiation and drug treatment. This kit detects cell apoptosis, which takes a short time. It only needs one step of staining reaction, and it can be detected after washing.

Notes
1. Before use, please centrifuge the product to the bottom of the tube briefly, and then perform subsequent experiments.
2. When using components A and B, please wear masks and gloves, and if they touch the skin, please rinse immediately with plenty of water.
3. Fluorescent dyes all have quenching problems, please avoid light as much as possible to slow down fluorescence quenching.
4. For your safety and health, please wear a lab coat and disposable gloves.

Instructions:

UE-6063

Asked questions:

◆为什么出现了一些非特异标记？
1.有些细胞或组织，比如平滑肌，nuclease或polymerase的酶活性水平较高，易导致假阳性，出现非特异性的荧光标记。因此，取出细胞或组织后要立即固定并充分固定，以阻止这些酶的活性；
2.使用了不恰当的固定液，推荐使用4%多聚甲醛；
3.TUNEL反应时间过长，或者在反应中细胞或组织表面未能一直保持湿润。因此。要注意控制反应时间，并确保反应液能很好地覆盖样品。
4.石蜡、脂肪、血液、体液等都较易与染料结合，所以组织切片制作时要保持干净、脱蜡要彻底；
5.有些试剂或细胞存在自发荧光，选择染料时要避开自发荧光的颜色。

◆为什么没有染上荧光？
1.固定不充分。固定液首选4%多聚甲醛，现用现配。不建议使用乙醇，乙醇固定液对组织的渗透力较弱，影响TUNEL标记效率；
2.通透不充分。细胞与冰冻切片，可用0.2% Triton X-100溶液通透5-30min，石蜡切片推荐使用蛋白酶K，37℃通透30 min；不同的细胞和组织所需要的通透时间略有不同，可以适当调整；
3.延长标记时间至2h，并适当增加TdT酶及dUTP的用量；
4.确定实验对象细胞凋亡时有DNA断裂现象发生。

◆为什么假阳性过高？
1.阴性对照有染色，则说明是非特异性染色；
2.若阴性对照无染色，那么可能是由于固定不充分，组织内有内源性核酸酶的残留，使得DNA全部被切断呈现阳性染色。

◆如何对细胞核进行复染？
可在TUNEL反应结束后对细胞核进行染色。

◆为什么荧光背景高？
1.支原体污染：可以使用支原体染色检测试剂盒验证；
2.TUNEL反应过强：可以用试剂盒提供的TdT酶稀释液将TdT酶稀释2-5倍后再按照说明书操作，稀释后的TdT酶仅供当日使用。

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