Description
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Transfection Reagent for Astrocytes (Primary Astrocyte Cells, Astroglia)
- Proprietary cationic lipids formulation
- High transfection efficiency of small RNA (siRNA, shRNA, miRNA), mRNA, pDNA
- Effective and robust intracellular delivery
- Kit includes Transfection Enhancer reagent
- Produces consistent results, lot-to-lot, plate-to-plate, and well-to-well
- Work in the presence of serum
- A proven reagent for establishing stable cell lines
- Optimized transfection protocols are adapted for use with both standard & reverse transfection methods
- Download in vitro Astrocyte transfection protocol: [PDF]
- Download Astrocyte CRISPR/Cas9 transfection protocol: [PDF]
- Download PowerPoint presentation for Astrocyte cells transfection kit: [PPT]
- Developed and manufactured by Altogen Biosystems
Transfection Efficiency:
Reagent exhibits at least 70% transfection efficiency of siRNA delivery. Transfection efficiency was determined by qRT-PCR.
Transfection Protocol and MSDS:
Download Altogen Biosystems Astrocyte Transfection Protocol: [PDF]
Download MSDS: [PDF]
Astrocyte Cells (Primary Astrocytes and Astroglia):
The human brain comprises two major cell types: neurons and glia. Neurons are responsible for transmitting nerve signals while glia safeguard neurons. Astrocytes, a sort of glial cells, make over 50% of all brain cells. In the event of any distress to the Central Nervous System (CNS), a process called gliosis is triggered. Gliosis involves the rapid differentiation of glial cells to provide mechanical support to neurons. However, excessive gliosis is symptomatic of various forms of brain cancer. Per the study published in the January 2016 issue of Tumor Biology, astrocytes play an important role in controlling the environment around a tumor. Astrocytes retard the detection of cancer cells by the immune system and help enhance their proliferation. Preclinical research targeting astrocytes can provide valuable insight into molecular pathways that can curb tumors. Transfection is a well-regarded technique to conduct preclinical research in many different cells, including astrocytes.
Astrocytes are star-shaped glial cells in the brain and spinal cord. Astrocytes are supportive cells, and their functions include biochemical support of endothelial cells, which form the blood-brain barrier, providing nutrients to the nerve tissue, extracellular K+ level regulation, neurotransmitters removal, and helping repair the brain and spinal cord following traumatic injuries. They are also dependent on myelination and synapses formation. Many primary astrocytes express the intermediate filament glial fibrillary acidic protein or GFAP. Research indicates that astrocytes communicate with neurons through the release of gliotransmitters via a calcium ion process. There are three types of astrocytes in the central nervous system: fibrous, protoplasmic and radial. It is believed astrocytes cell bodies are separate and do not overlap.
Astrocytes, also known as astroglia, are cells in living organisms that make up parts of the nervous system, particularly in the brain and spinal cord. They work with the body’s natural response to trauma in the brain and spinal cord, by repairing and providing nutrients to affected areas. Because of this, astrocytes may be useful in the study of specific medical treatments for patients suffering from spinal cord injuries. These cells are crucial to research in the field of neuroscience.
Primary cell cultures are used in biological and gene therapy studies and serve as valuable model systems that may more accurately represent the biology of healthy cells. Many cultured cell lines, as well as the majority of primary cell cultures, can be transfected with exogenous nucleic acids when appropriate transfection approaches are employed. Since the majority of transfection methods causes significant toxicity in primary cell cultures, optimizing this procedure, specifically the protocol and reagents to be utilized, is essential for developing an effective transfection strategies for a given cell type. ALTOGEN® Kits for primary cells and sensitive cell lines have been designed to have significantly lower cytotoxicity than other alternatives.
Data:
Figure 1. SiRNAs targeting Lamin A/C mRNA or non-silencing control siRNA were transfected into actrocyte cells following the recommended protocol. At 48 hours post-transfection the cells were analyzed by qRT-PCR for gene expression levels. 18S rRNA levels were used to normalize the Lamin A/C data. Values are normalized to untreated sample. Data are means ± SD (n=3).
Figure 2. Protein expression of Lamin A/C in Astrocyte cells. DNA plasmid expressing Lamin A/C or siRNA targeting Lamin A/C were transfected into Astrocyte cells following Altogen Biosystems transfection protocol. At 72 hours post-transfection the cells were analyzed by Western Blot for protein expression levels (normalized by total protein, 10 µg of total protein loaded per each well). Untreated cells used as a negative control.
Selected Astrocyte Transfection Reagent product citations:
- Journal of Neuroscience. 2013 33(44). Foxo3a Transcriptionally Upregulates AQP4 and Induces … Kapoor et al [PDF]
- Mol Cell Biol. 2013 33(7). SCO2 induces p53-mediated apoptosis by Thr845 phosphorylation … Madan et al [PDF]
Astrocyte Transfection Reagent
Altogen Biosystems:
Altogen Biosystems provides pre-optimized transfection kits and electroporation products for life sciences and cancer research. Transfection protocols are optimized for individual cancer cell lines. Altogen Biosystems developed two types of in vivo delivery kits (animal transfection): 1) Tissue-targeted reagents (delivery of proteins, DNA, and RNA into liver, pancreas, or kidney tissues), and 2) Broad range in vivo biodistribution reagents (PEG-Liposome based reagent, Nanoparticle-based in vivo reagent, Lipid-based transfection kit, and Polymer-based kit). Advanced formulation of reagents and optimized transfection protocols provide efficient intracellular delivery of proteins, DNA, mRNA, shRNA, siRNA, and other negatively charged biomolecules in vitro and in vivo. Read more about transfection technology at Altogen’s Transfection Resource.
Altogen Research Services:
Altogen Labs provides GLP-compliant contract research studies for pre-clinical research, IND applications, and drug development. Biology CRO services include: Xenograft models (30+), development of stable cell lines, ELISA assay development, cell-based and tissue targeted RNAi studies, safety pharm/tox assays, and other studies (visit AltogenLabs.com).
Volume Options:
- 0.5 ml (Catalog #1711)
- 1.5 ml (Catalog #1712)
- 1.5 ml CRISPR (Catalog #2110)
- 8.0 ml (Catalog #1713)
AltogenBiosystems是一家开发和制造用于生命科学研究,药物发现和开发的转染试剂盒的生物技术公司。转染试剂盒针对特定癌细胞系和原代细胞培养进行了优化,可将生物分子有效递送到靶组织中。通过先进的试剂配方和优化的转染方案实现体外(癌细胞系)和体内(动物组织靶向试剂、癌细胞系)递送货物分子,包括质粒DNA,各种类型的RNA(mRNA,siRNA,shRNA,microRNA),蛋白质和小分子研究。
Altogen生命科学公司致力于研发,生产和销售特定细胞系的转染试剂,用于细胞间生物分子的传递,并通过对转染试剂类型的设计将siRNA和质粒DNA有效地转入不同的细胞系和原代细胞内。Altogen公司开发的聚合物,脂质体,纳米粒子为基础的转染技术分别针对分子生物学,组合化学,和细胞生物学而分别应用。Altogen定制服务提供符合GLP要求定制研究服务,包括代稳定的细胞系,细胞银行和冷冻保存,焦磷酸测序,克隆,RNA干扰(RNAi)和基因沉默服务,发展分析,siRNA文库筛选,并转染服务。稳定的肿瘤细胞株和原代细胞的产生,可以是非常昂贵和费时。该公司的细胞培养科学家的细胞株的选择,无论是利息或shRNA表达载体的稳定表达的基因改造。标准的RNAi技术服务,包括设计与合成的siRNA的利益,验证siRNA的沉默效率,siRNA转染条件的优化,使高效的基因沉默细胞系或原代培养细胞的靶基因。转染培养细胞的瞬时或稳定的引入外源性分子和遗传物质(即RNA或DNA),通常是在生物实验室用来研究基因功能,基因表达的调节,生化映射,突变分析,和蛋白质的生产。科学家利用各种载体分子,这种分子,使质粒DNA(PDNA),信使RNA(mRNA),短干扰RNA(siRNA),小分子RNA(miRNA)的,并进入肿瘤细胞株和原代细胞的蛋白质的基因交付。不幸的是,无单提货的方法或转染试剂,可以适用于所有类型的细胞,细胞的细胞毒性和转染效率显着不同,取决于试剂,协议,并正在利用细胞类型。Altogen生物系统公司提供超过60种类型的细胞的预优化转染试剂盒。纳米粒子,脂质和聚合物基ALTOGEN®在体内转染试剂,使交付功能的RNA和DNA分子在体内。PEG脂质体在体内输送系统减少由于PEG修饰的先天免疫反应,并提供高效的siRNA转染的DNA,并在体内的蛋白质。由科学“杂志(2010年12月17日):PEG脂质体在体内转染试剂盒siRNA的特色Altogen生物系统功能的特定细胞系转染试剂盒
120+细胞转染试剂和活体组织靶向试剂盒制造商AltogenBiosystems是一家生物技术公司,开发和制造用于生命科学研究、药物发现和开发的转染试剂盒。Altogen®体内转染试剂可有效地将生物分子导入靶组织。细胞转染试剂盒针对特定的癌细胞系和原代细胞进行了优化。通过先进的试剂配方和优化的转染方案实现货物分子(DNA、RNA、蛋白质)的高效传递。AltogenBiosystems利用高分子化学、分子和细胞生物学的专业知识,开发了新的体内外给药技术。转染是将外源分子导入培养细胞中,常用于研究基因功能、基因表达调控、生化定位和蛋白质生产。不幸的是,由于细胞毒性和转染效率的差异很大,并且取决于所使用的试剂、方案和细胞类型,因此没有一种单一的传递方法或转染试剂可应用于所有类型的细胞。AltogenBiosystems为120多个癌细胞系和原代细胞类型提供优化的转染试剂盒和电穿孔产品。体内转染试剂可实现组织靶向给药。Altogen的转染试剂盒包括用于体外(癌细胞系)和体内(用于动物研究的组织靶向试剂)转染的转染增强剂试剂和转染复合物冷凝器。Altogen实验室提供符合GLP的实验室合同研究服务。我们的生物CRO服务包括异种移植物的疗效、IND应用的pharm/tox研究和安全性测试、分析开发(ELISA、IC-50、qPCR)、90多个异种移植物动物模型、RNAi和基因沉默服务。Altogen的细胞培养科学家通过在28天内培育出稳定的细胞系,将选择的细胞系转化为稳定表达感兴趣的基因。
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DXY721认为:
悬浮细胞和贴壁细胞在转染过程中差别不大,主要差别在于转染后的筛选,当然如果你做的是瞬时转染就不存在筛选的问题了。
其实转染的过程很简单,问题是能不能转的进去的,转染率能有多少,转进去是否可以稳定表达目的蛋白等等。
我们也是用脂质体做悬浮细胞的转染,说明书上都有具体的操作过程,将脂质体和目的基因按比例混合,然后加到细胞悬液里就OK了,说的简单,实际上还是有一些细节要注意的,比如脂质体和目的基因混合的比例,转染的细胞数,细胞的代数,细胞的状态,有的还要求在转染的前一天传代一次,不过不要怕,这些在脂质体说明书上都有明确的说明,按照说明书做就可以了。
jinghuanlv认为:
悬浮细胞和贴壁细胞转染还是有很大不同的。
脂质体转染的原理基于电荷吸引原理,先形成脂质体-DNA复合物,散布在细胞周围,然后通过细胞的内吞作用,将目的基因导入细胞内,而脂质体复合物与贴壁细胞的接触机会比悬浮细胞高出很多倍,所以,脂质体转染时悬浮细胞的转染效率要明显低于贴壁细胞。
我们实验室转染悬浮细胞是用的电穿孔法,目前为止,悬浮细胞转染的最好方法还是电转,我们实验室用的电转仪是Bio-Rad的,使用条件是电压250V,电容975uF,效果不错,不妨一用。
本人研究生
我转的是7901、7901/DDP两种细胞,前者7901细胞很容易就转上,并且转后,状态良好,可是7901/DDP一转就死,我用的是吉玛慢病毒,转24小时后换液,刚开始一两天,没有异常,但后来细胞慢慢就死了,并且不是漂浮的,很多是贴着壁死,像是瓦解了一样
这是未转时细胞的样子
这是细胞转后,死亡的样子
并且即使是有些细胞未死,细胞后来也变得很脏,感觉有很破碎的细胞碎片
本人实验小白,**园子里大神指点,急,实在不知道怎么回事
GFP发出绿色荧光的原理是Ca离子进入GFP的beta-barrel结构中引起的特定能级,因此只要这个结构仍然保持着,就可以发出荧光。
由于GFP的beta-barrel结构非常稳定,一些版本的GFP蛋白(如EGFP)甚至能抵抗94C的高温几分钟而不完全变性,因此想在溶液状态下去掉GFP的荧光是很难的,一般需要用光漂白法。
基于其非常稳定的结构,即便细胞被固定了,仍然会有一部分的GFP蛋白保持其构象而发出荧光。此时荧光可能较弱。在荧光显微镜下是有可能看得到的。
影响转染试验的因素:
1转染试剂跟细胞系不匹配
转染试剂跟细胞系也是讲究配合默契的,使用同一种试剂,不同细胞系转染效率通常不同。但细胞系的选择通常是根据实验的需要,因此在转染实验前应根据实验要求和细胞特性选择适合的转染试剂。每种转染试剂都会提供一些已经成功转染的细胞株列表和文献,通过这些资料可选择最适合实验设计的转染试剂。当然,最适合的是高效、低毒、方便、廉价的转染试剂。
2细胞状态变化
因为有些细胞系是不稳定的,可能随着培养时间的改变,培养条件的不同,不同的选择压力,可能引起不同的克隆选择。因此就算是同一个细胞系,在不同条件下转染能力的差异可能会
很大
(1)转染试剂与细胞不匹配
细胞转染最适合的不是原代细胞,也不是传代很多次的细胞。这是因为细胞培养在实验室中保存数月和数年后会经历突变,总染色体重组或基因调控变化等而演化。这会导致和转染相关的细胞行为的变化。最适合转染的细胞是经过几次传代后达到对数生长期的细胞,细胞生长旺盛,最容易转染。
(2)把握时机
没错!转染也有适当的时机,相比较非分裂细胞——分裂细胞往往要比静止细胞更易于摄取并表达外源DNA。因此对大多数转染操作而言,细胞都在转染当天或前一天种板。
同样重要的是细胞在种板进行转染时不应处于过度生长的状态,如癌细胞数量过多,互相叠加,营养物质耗竭,代谢废物积聚,转染率低下也是很正常的!
因此,一定要在最适细胞密度时转染,才能获得较高的转染率。不同的转染试剂,要求转染时的最适细胞密度各不相同,即使同一种试剂,也会因不同的细胞类型或应用而异。
(3)微生物来捣乱
培养物可被细菌、酵母、真菌、病毒、支原体、甚至其他细胞种类所污染。各种污染都会导致产生错误的结果。
(4)交叉污染
如果同一个实验室同时培养不同种类的细胞,很同意发生“细胞串门”的现象,造成交叉污染。
3转染方法
不同转染试剂有不同的转染方法,但大多大同小异。转染时应跟据具体转染试剂推荐的方法,但也要注意,因不同实验室培养的细胞性质不同,质粒定量差异,操作手法上的差异等,其转染效果可能不同,应根据实验室的具体条件来确定最佳转染条件。
(1)血清
转染后未及时加入血清,会导致细胞大量死亡。一般要在转染后的4-6小时换液且换为有血清的培养基。也可以在原来的无血清培养基里面滴加血清。这个时候,最好不要换液,不要打扰细胞,让它安安静静地休息。但是也不能过早加入血清。过早的话,会引起未转染的细胞疯狂生长。那么,什么是最佳时机呢?在20%的细胞变圆的时候,就是加血清的最佳时机。
不过要特别注意:血清是一种包含生长因子及其它辅助因子的不确切成分的添加物,对不同细胞的生长作用有很大的差别。血清质量的变化直接影响细胞生长,因此也会影响转染效率。新加培养基的预热对细胞转染很有帮助。
(2)DNA质量
DNA质量对转染效率影响非常大。一般的转染技术(如脂质体等)基于电荷吸引原理,如果DNA不纯,如带少量的盐离子,蛋白,代谢物污染都会显著影响转染复合物的有效形成及转染的进行。
4载体构建
转染载体的构建(病毒载体,质粒DNA,RNA,PCR产物,寡核苷酸等)也影响转染结果。因此选择组成或可调控,强度合适的启动子也很重要,同时做空载体及其它基因的相同载体构建的转染正对照可排除毒性影响的干扰。
所以转染用的质粒首先要保证数量,一般为2μg以上。质粒纯度不够或者含有细菌LPS或其他对细胞有毒害作用的物质,也会影响转染效率。这个时候,就应该对质粒进行纯化和浓缩。
以上就是细胞转染率低的主要原因了,在实验过程中有没有遇到什么棘手的问题呢,欢迎留言讨论
做的比较好的,一般都是上海地区的,你可以看下基尔顿生物。
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