
Cell Line Information:
Designations: 293 [HEK-293]Depositors: FL GrahamGrowth Properties: adherentOrganism: Homo sapiensMorphology: epithelialSource:Organ: embryonic kidneyApplications: efficacy testing, transfection host, viruscide testingReceptors: vitronectinTumorigenic: YESAge: fetusComments: The cells containes Adenovirus 5 DNA from left ends of the viral genome. The line is excellent for titrating human adenoviruses. The cells express an unusual cell surface receptor for vitronectin composed of the integrin beta-1 subunit and the vitronectin receptor alpha-v subunit. The Ad5 insert was cloned and sequenced, and it was determined that a colinear seqment from nts 1 to 4344 is integrated into chromosome 19 (19q13.2)
The Transfection Reagent:
293 Cell Avalanche™ Transfection Reagent is a new, proprietary solution specifically designed for transfection on 293 Cells. The proprietary formulation of lipids and polymersensures the highest possible transfection efficiencies and viabilities for 293 Cells. No other transfection reagents can match the efficiency, convenience, and gentleness of 293 Cell Avalanche™ Transfection Reagent for transfection on 293 Cells. For details of the developing process of this reagent, please go to: How did EZ Biosystems develop Avalanche™ Cell type/cell line specific transfection reagent series?
References:
Amini M, Wang H, Belkacemi A, Jung M, Bertl A, etal.Identification of Inhibitory Ca2+ Binding Sites in the Upper Vestibule of the Yeast Vacuolar TRP Channel. iScience. 2019 Jan 25;11:1-12. PubMed PMID: 30572205; PubMed Central PMCID: PMC6299153.
Features:
- Specifically optimized to deliver nucleic acids into 293 Cells
- Highest efficiency to ensure experimental success
- Lowest Cellular Toxicity-maintain cell density and reduce experimental biases
- 0.5 ml is able to transfect about 1000 wells of 24-well plate
- Simplest and straightforward protocols
- Deliver single or multiple plasmids
- Compatible with serum
- Suitable for Reverse Transfection
- Reproducible: due to highly controlled chemical synthesis of each of the ingredients, the reagent forms uniformly sized complex particles with nucleic acids. With optimized protocol, our reagent will ensure the reproducible highest transfection results.
- Economical: High efficiency means less amount of nucleic acid & reagent is needed
- Developed and manufactured by EZ Biosystems
EZBiosystems美国EZBiosystems™公司是一家全球性转染试剂和基因表达生产服务提供商,公司的研发宗旨是让生命科学研究变得更简单,快速,容易!该公司主要产品之一为新一代转染试剂Avalanche 系列产品,该产品集合了组合化学,分子生物学和细胞生物学技术。公司致力于开发针对不同细胞类型的专用转染试剂。公司研发团队由多个学科经验丰富的专家组成,包括组合化学、脂类化学、生物物理学、分子生物学和细胞生物学。目前,EZBiosystems™主要提供广谱性基因及siRNA体外转染试剂、体内转染试剂、生物治疗性蛋白表达专用转染试剂以及150余种细胞特异性转染试剂。
EZBiosystems特约代理EZBiosystems品牌名称:EZBiosystemsEZBiosystems品牌logo:EZBiosystems品牌官网:http://ezbiosystems.com/EZBiosystems品牌链接:http://www.annoron.com/brand-342.htmlEZBiosystems品牌介绍:EZ生物系统™是一家全球性的基因转染和基因表达产品和服务提供商。EZBiosystems™isaworldwideprovideroftransfectionandgeneexpressionproducts&services.OneofthemajorfocusesofEZBiosystems™hasbeentheresearch,development,andmanufactureofnext-generationtransfectionreagents----theAvalanche®TransfectionReagentseriesbyapplyingourexpertiseincombinatorialchemistry,molecularBIOLOGy,andcellbiology. EZBiosystems品牌相关产品:品牌货号名称规格EZ BiosystemsEZT-CRPR-1Avalanche®-CRISPR Transfection Reagent0.5ml 1.5mlEZ BiosystemsEZT-OMNI-1Avalanche®-Omni Transfection Reagent0.75ml 1.5ml5*1.5mlEZ BiosystemsEZT-EVDY-1Avalanche®-Everyday Transfection Reagent0.75ml 1.5ml
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荧光标记物常用的有几十种,比如FITC, PE等等,各个生产厂家还有自己的专利产品
探针
ddH2O36.25ul
buffer(瓶3)5ul
核苷酸混合液(含DIG-dUTP)(瓶2)2.5ul
dNTPStockSolution(瓶4)2.5ul
引物F1ul(10pmol/ul)
R1ul(10pmol/ul)
酶(瓶1)0.75ul
DNA1ul(100pg)
程序:35个循环,退火温度55度。
第一次通过探针标记得到了相应的PCR标记产物,亮度和对照相当,片段大小比标记的探针模板大,证明整个过程应该没有问题。
但一个月后重新用相同的体系,相同的程序,只是探针模板的量不同却什么都扩不出来。不知道问题出在哪了!
TUNEL的缺点是:1),操作要求高,组织样本需要固定(即使是培养细胞,也需要固定),不恰当的固定方法对实验结果影响很大,导致背景过高或者信号过弱,因此实验结果重复性不好;有的人花上半年做一个体内的TUNEL是一点都不奇怪的。2),大部分诱导凋亡的药物也引起DNA损伤,从而产生DNA断裂,易引入假阳性;3)凋亡晚期细胞基因组大量降解,导致TUNEL标记反而减少,因此此法反应的是早期凋亡比例,不能严格反应凋亡比例,属于半定量研究。
尽管有如此多缺点,但由于其是目前为数不多的能原位标记凋亡细胞的方法,因此用于组织体内凋亡研究仍然是首选。但体外细胞实验研究,很少用此法。
凋亡检测中,TUNEL并不是过时的方法,现在研究凋亡的文献仍然常见。而且相反,还比以前多一点,因为现在体内实验越来越多了,甚至线虫的凋亡研究,都用TUNEL。
凋亡研究中,的确有几种方法过时了,当然是因为有替代方案了,比如DNAladder,电镜。至于AnnexinV是不能用来和TUNEL一起评论的,前者用于细胞,而且主要是悬浮细胞,后者主要用于组织。虽然有人也做镜下的AnnexinV观察,TUNEL的细胞staining,但这都不是主流。
回到lz的原帖,的确如大家所言,做贴壁细胞,不推荐用TUNEL。如果是经典凋亡途径,只是确定比例,用最经典,最常用的subG1法即可,如果是不确定是否是凋亡,用AnnexinV,不过贴壁细胞用此法,要注意消化时间。
那为什么SFDA不批准CA199CEAAFP等检测试剂盒作为癌症检测的手段呢?
想请问下那个公司有专门的蛋白质荧光标记试剂盒出售。最好的是CY5的,我准备做三标。性价比越高越好
那些做过的前辈们指导一下。

