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View additional product information for Vitronectin (VTN-N) Recombinant Human Protein, Truncated - FAQs (A14700, A31804)
37 product FAQs found
可以。在mTeSR培养基+BD Matrigel基底膜基质条件下培养并冻存的PSCs细胞可复苏至Gibco Essential 8培养基中并种植于VTN-N基质上。某些细胞系复苏至冻存前的生长培养基及基质中可能会效果更好。在随后的下一次传代过程中，就可使用EDTA将这些细胞传代至Gibco Essential 8培养基+VTN-N的组合中了。
我们推荐您使用Gibco Geltrex 无LDEV，经hESC验证的低生长因子基底膜基质（货号A1413301或A1413302）或在不含异源成份的应用中使用Gibco玻连蛋白（VTN-N）-截短型重组人源蛋白（货号A14700）。
分散酶与胶原酶一类的酶并不适用于Gibco Essential 8培养基和Gibco玻连蛋白（VTN-N）的组合。使用这些酶类传代会导致细胞活力和贴壁能力下降。
培养在Gibco Essential 8培养基与VTN-N组合中的细胞应使用EDTA进行传代。
您预期可看到正常的多能干细胞（PSC）形态。PSC的预期生长形态是如图所示的典型形态为，包括边界清晰的紧密聚集克隆和高核质比。
培养于其它无饲养层培养体系的细胞，例如mTeSR培养基搭配Matrigel基底膜基质，或StemPro hESC SFM搭配Geltrex基质，也能够成功培养于Gibco Essential 8培养基搭配VTN-N的体系中。此外，在饲养层细胞搭配KnockOut SR中培养的PSC经证明也能够培养于Gibco Essential 8培养基搭配VTN-N的组合中。不过在更换培养基体系之前，细胞必须手工传代或用EDTA进行传代到Gibco Essential 8培养基搭配VTN-N的体系中。
Gibco Essential 8培养基与玻连蛋白经证明能够支持PSC的生长达50代以上而不会发生任何核型异常，同时这些PSC仍保持着分化为三胚层的能力。James Thomson实验室Chen等报道（Chen G, Gulbranson DR, Hou Z et al (2010) Chemically defined conditions for human iPSC derivation and culture.Nat Methods 8:424–429.），玻连蛋白的VTN-N变异体与Gibco Essential 8培养基联用时相比野生型玻连蛋白能更好地支持人多能干细胞的贴壁和存活。。
是的。VTN-N是成份明确的人重组蛋白。
以Gibco玻连蛋白（VTN-N）作为细胞生长基质使用Gibco Essential 8培养基的条件相比使用其他无饲养层系统而言，有三个主要差别需要加以考量：
•通常细胞的传代时间比其他无饲养层培养基提前24小时左右。
•细胞在达到85%左右的汇合度时即需要传代。如果细胞传代时超过了85%的汇合度，细胞健康度及最终的细胞得率都会下降。
•细胞应使用EDTA进行传代。不推荐使用胶原酶或分散酶。
由于VTN-N是成份明确的人源重组蛋白，因此相比人血浆来源的玻连蛋白和标准基底膜抽提物（BME）而言，能够有助于降低PSC培养的变异度。此外，VTN-N与全长的玻连蛋白及其他成份明确的基质相比更加经济和便于缩放PSC培养体系。
Gibco玻连蛋白（VTN-N）是按照人源玻连蛋白62-478号氨基酸片段设计，在大肠杆菌中表达的截短型人源重组蛋白。VTN-N经内涵体纯化和重新折叠，适合在人源PSC无饲养层培养体系作为基质使用（Chen G, Gulbranson DR, Hou Z et al (2010) Chemically defined conditions for human iPSC derivation and culture.Nat Methods 8:424–429）。当与Gibco Essential 8培养基联用时，VTN-N经验证能够帮助维持多种PSC细胞系的多能性和正常生长。
如果您按照我们产品插页中的描述进行了测试，但未见到任何反应，则可能由以下几种原因导致：
- 蛋白未按照说明书推荐方式进行溶解。
- 溶解后的蛋白放置时间过长或蛋白发生沉淀。我们推荐您在重新溶解后的3-6个月内使用这些蛋白产品。
- 当重新溶解的蛋白溶液浓度低于0.1 mg/mL时，未添加载体蛋白。低于0.1 mg/mL的工作液应立即使用；我们不推荐长期储存此浓度的蛋白溶液。
- 蛋白溶液经过多次冻融循环或暴露于高温条件下。
- 使用错误类型的容器操作蛋白（某些蛋白非常容易粘附于某些塑料器皿上）。
生物分析法常被用来检测某一特定生长因子或细胞因子的生物学活性。在大多数情况下，研究人员一般基于各类指示性细胞——如某些原代细胞或细胞系——来开展生物分析。最为常用的生物分析法包括细胞增殖分析(https://www.thermofisher.com/content/dam/LifeTech/Documents/PDFs/Cell-Proliferation-Assay.pdf)，细胞趋化性分析(https://www.thermofisher.com/content/dam/LifeTech/Documents/PDFs/Chemotaxis-Assay.pdf)，细胞因子生成分析(https://www.thermofisher.com/content/dam/LifeTech/Documents/PDFs/Cytokine-Production-Assay.pdf)和细胞毒性分析(https://www.thermofisher.com/content/dam/LifeTech/Documents/PDFs/Cytotoxicity-Assay.pdf)。通常以ED50来表示某一细胞因子的生物活性，该值代表了能够诱发50%最大效应的细胞因子浓度。这一表示效价的方法仅适用于那些剂量效应曲线为S形的细胞因子。
Thermo Fisher Scientific所提供的重组蛋白产品通常在大肠杆菌，昆虫细胞或哺乳动物细胞等不同表达系统中产生。不同表达系统之间产生的重组蛋白的主要差别在于这些蛋白所携带的翻译后修饰，如糖基化修饰。在大肠杆菌中生成的重组蛋白未经糖基化。在昆虫细胞中产生的重组蛋白通常有部分糖基化，但不含半乳糖和唾液酸，糖链也无复杂分支。在哺乳动物细胞中产生的重组蛋白会带有完全的糖基化修饰。
备注：Mimic Sf9 昆虫细胞（来源于sf9昆虫细胞系，经改造可稳定表达多种哺乳动物糖基转移酶）可用于表达带末端唾液酸和半乳糖的复杂的N-糖链。
反复冻融会对重组蛋白的稳定性造成影响。举例来说，冰冻会显著影响蛋白溶液的pH值，可能会导致蛋白变性（（Arch Biochem Biophys 384:398 (2000)）。
Gibco 玻连蛋白（VTN-N）-截短型重组人源蛋白（Gibco Vitronectin (VTN-N) Recombinant Human Protein, Truncated，货号 A14700）与Gibco Geltrex 无LDEV，经hESC验证的低生长因子基底膜基质（Gibco Geltrex LDEV-Free, hESC Qualified Reduced Growth Factor Basement Membrane Matrix，货号A1413301）两者均适用于Gibco Essential 8 Flex培养基。
We recommend using Vitronectin (VTN-N) Recombinant Human Protein, Truncated (Cat. No. A14700), but Geltrex LDEV-Free hESC-qualified Reduced Growth Factor Basement Membrane Matrix (Cat. No. A1413301 or Cat. No. A1413302) can also be used.
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These two proteins are the same but have different protein concentrations due to the different methods used to measure the protein concentrations.
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We have evaluated the use of Cytotune-iPS 2.0 Sendai Reprogramming Kit (Cat. No. A16517) for somatic cell reprogramming of CD34+ blood cells. For CD34+ cells, follow the instructions provided in the Cytotune 2.0 reprogramming manual for feeder-free reprogramming (pgs. 39-44). On Day 3, you can utilize rhVTN-N (Cat. No. A14700), Geltrex (Cat. No. A1413302), or rhLaminin-521 (Cat. No. A29248 or A29249). From Day 8 onward, rather than feeding daily with Essential 8 Medium, reprogrammed CD34+ cells should be fed every-other-day with StemFlex Medium.
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We have evaluated the use of Cytotune-iPS 2.0 Sendai Reprogramming Kit (Cat. No. A16517) for somatic cell reprogramming of both neonatal and adult human dermal fibroblasts. For fibroblasts, follow the instructions provided in the Cytotune 2.0 reprogramming manual for feeder-free reprogramming (pgs. 16-20). On Day 7, you may use rhVTN-N (Cat. No. A14700), Geltrex matrix (Cat. No. A1413302), or rhLaminin-521 (Cat. No. A29248 or A29249). From Day 8 onward, rather than feeding daily with Essential 8 Medium, we recommend that you feed reprogrammed fibroblasts every-other-day with StemFlex Medium.
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Yes. We recommend following the coating instructions for Vitronectin or rhLaminin-521 and using the culture and passaging recommendations in the StemFlex protocol.
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We recommed coating the plate at room temperature for 1 hour.
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This product should be thawed at room temperature for 10-15 minutes. It should not be thawed at 4 degrees C overnight which might result in poor product performance.
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The truncated form of vitronectin supports human pluriportent stem cells attachment and survival better than wild-type vitronectin. This truncated form has been optimized for use with Essential 8 Medium.
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Yes. PSCs cryopreserved from cultures of mTeSR Medium and BD Matrigel Basement Membrane Matrix may be thawed into Gibco Essential 8 Medium and plated on VTN-N. Certain lines may benefit from thawing into the medium and substrate they were growing in at the time of cryopreservation. Then at the next passage, use EDTA to passage the cells into Gibco Essential 8 Medium and VTN-N.
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We recommend using Gibco Geltrex LDEV-Free, hESC Qualified Reduced Growth Factor Basement Membrane Matrix (Cat. No. A1413301 or A1413302) or Gibco Vitronectin (VTN-N) Recombinant Human Protein, Truncated (Cat. No. A14700) for xeno-free applications.
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Enzymes such as dispase and collagenase do not work well with cells cultured in Gibco Essential 8 Medium on VTN-N. Use of these enzymes for passaging cells results in compromised viability and attachment.
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Cells cultured in Gibco Essential 8 Medium and VTN-N need to be passaged with EDTA.
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You should expect to see normal pluripotent stem cell (PSC) morphology. The expected morphology of PSCs is demonstrated specifically by tightly packed colonies with defined borders and a high nucleus-to-cytoplasm ratio.
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Cells cultured in other feeder-free media systems, such as mTeSR Medium with Matrigel Basement Membrane Matrix, or StemPro hESC SFM with Geltrex Matrix, can be successfully cultured in Gibco Essential 8 Medium and VTN-N. In addition, PSCs grown on feeders with KnockOut SR have also been shown to be successfully cultured in Gibco Essential 8 Medium on VTN-N. However, when changing media systems, cells must be passaged either manually, or with EDTA prior to culturing in Gibco Essential 8 Medium on VTN-N.
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Gibco Essential 8 Medium and vitronectin have been shown to support PSC growth for >50 passages without any signs of karyotypic abnormalities, and maintain the ability of PSCs to differentiate into all three germ line lineages. As published by Chen et al (http://www.ncbi.nlm.nih.gov/pubmed/21478862) in the laboratory of James Thomson, the VTN-N variant of vitronectin supports human pluripotent stem cell attachment and survival better than wild-type vitronectin when used in conjunction with Gibco Essential 8 Medium.
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Yes. VTN-N is a defined, recombinant human protein.
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Here are three major differences to be taken into consideration when culturing cells in Gibco Essential 8 Medium on Gibco Vitronectin (VTN-N) compared to other feeder-free systems:
- Cells should be typically passaged ~24 hours sooner than they would be with other feeder-free media.
- Passaging should take place when cells are at ~85% confluency. If cells are passaged when they are more than 85% confluent, the health of the cells and final cell yield may be compromised.
- Cells must be passaged in EDTA. Collagenase and dispase are not recommended.
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Since VTN-N is a defined, recombinant human protein, variability is reduced in PSC cultures compared to human plasma-derived vitronectin and standard basement membrane extracts (BMEs). In addition, compared to full-length vitronectin and other defined substrates, VTN-N helps enable economical and scalable PSC culture.
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Gibco Vitronectin (VTN-N) is a recombinant, truncated human protein corresponding to the amino acid fragment 62-478 of human vitronectin expressed in E. coli. VTN-N is purified from inclusion bodies and refolded for use as a substrate for the feeder-free culture of human PSCs (http://www.ncbi.nlm.nih.gov/pubmed/21478862). When used with Gibco Essential 8 Medium, VTN-N has been proven to maintain pluripotency and normal growth characteristics in multiple PSC lines.
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Yes. Following 2 passages on the rhLaminin-521 matrix, Versene or EDTA passaging should be used to subculture PSCs.
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Both Gibco Vitronectin (VTN-N) Recombinant Human Protein, Truncated (Cat. No. A14700) and Gibco Geltrex LDEV-Free, hESC Qualified Reduced Growth Factor Basement Membrane Matrix (Can. No A1413301) have been successfully used with Essential 8 Flex Medium.
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