StemPro™ Neural Stem Cells - FAQs

NSC不会被神经元特异性的标志物所染色。如果发现细胞被染色，则可能是由于抗体的非特异性所导致的。我们推荐您对抗体特异性和染色方案进行检查：

•对第一抗体进行滴定，以找到最佳的抗体工作浓度。
•调整破膜方案，因为高浓度的破膜剂会导致高背景。
•使用足量的封闭液——如5-10%的血清——来封闭非特异性的着色。
•包含同型（抗体）对照。

这里列举了一些可能导致您培养细胞失败的原因：

•不正确地储存细胞：这些细胞应保存于液氮中。
•未使用推荐的培养基：Gibco H9来源的NSC（货号N7800）和GibcoStemPro NSC（货号A15654或A15655）两种产品所使用的完全型培养基具有不同的配方。StemPro NSC的培养基中需添加额外成份（肝素和抗坏血酸）。
•不正确地复苏细胞：请勿在37°C条件下化冻细胞超过2分钟。细胞一旦化冻，应立即移至50-mL离心管中，之后在不断摇动离心管的过程中逐滴加入（每秒一滴左右）预热的完全型培养基。
•化冻细胞后未对细胞活力进行计数和未正确种植细胞：您需要使用台盼蓝计数细胞活力。每管中的细胞至少达到1 x 10E6活性细胞/mL。对于H9来源的NSCs细胞而言，推荐以大于1 x 10E5活性细胞/cm2的密度进行种植。对于StemPro NSC细胞而言，推荐以大于7 x 10E4活性细胞/mL的密度进行（悬浮）培养。
•培养板未经正确包被：对于H9来源的NSCs进行贴壁培养而言，您需要依照包被说明书中的方案来应用GibcoGeltrex基质，纤连蛋白或L型多聚鸟氨酸/层粘连蛋白对组织培养板进行正确包被。对于StemPro NSC细胞而言，我们推荐您进行悬浮培养，因为贴壁培养可能会导致细胞分化。

我们提供两类人源NSCs：

•Gibco StemPro神经干细胞（货号A15654或A15655）：分离自人胚胎脑部皮层组织，并按照药品生产质量管理规范（GMP）进行生产。每批产品均源自同一主细胞库（相同供体），所以批次之间的变异度很小。细胞倍增时间在100小时左右。
•Gibco 人神经干细胞（源自H9）（货号 N7800）：以专利方法诱导H9人源ESC产生。细胞倍增时间为约40-50小时，并随传代次数增加而延长。

经测试，两类细胞在解冻后至少在三代之内均保有增殖和分化潜能。两者也都能分化为神经元，星形胶质细胞和少突胶质细胞。不过，StemPro神经干细胞建议使用悬浮培养，因为贴壁培养可能会引起分化，而人神经干细胞（H9来源）可适应悬浮培养与贴壁培养。

以下生长因子可用于NSC的扩增：重组EGF（货号PHG0314），重组bFGF（货号PHG0024)，和重组VEGF（货号PHC9394）。此外，包括BDNF在内的多几种神经营养因子例如BDNF（货号10908010），CNTF（货号PHC7015），和GDNF（货号PHC7044）也可适用于相关研究。

人NSC可培养于Gibco StemPro NSC SFM（货号A1050901）和Gibco Geltrex 基质/或Gibco CELLstart 底物基质预先包被的培养皿中。另外，如果用户的研究目的是获得神经元，也可在已添加了Gibco B-27添加剂（不含维生素A）的Neurobasal培养基（不含维生素A） + 预先包被的培养皿中培养NSC。

当按照克隆密度进行培养时，通常可基于用神经球的形成能力来鉴定NSC（Nat Methods 2:333 (2005)）。也可通过（1）Sox1，Sox2和Nestin的RT-PCR检测或（2）nestin，Pax6，Sox2和Ki67的免疫组化染色来鉴定NSC。

神经干细胞（NSC）是神经系统中能够自我更新的多专能细胞，拥有分化为神经元，少突胶质细胞和星形胶质细胞的潜能。神经干细胞可由胚胎干细胞诱导分化而来，或从皮层，脑室下区（SVZ）和脑室区等多个不同脑区分离获得，或从骨髓来源的间充质干细胞（MSC）生成 (J Cell Biochem 114:764 (2013))。NSC是研究神经发生和神经递质与受体功能的宝贵工具。来自在各类动物疾病模型中的NSC被广泛应用于包括例如帕金森氏病与亨廷顿氏病舞蹈症在内的等多种不同类型的CNS疾病的研究中（J Cell Biochem 114:764 (2013)）。

NSCs do not stain with neuron-specific markers. If staining was observed, this could have resulted from antibody non-specific staining. We recommend checking antibody specificity and the staining protocol:

- Titrate primary antibody to find the optimal antibody working concentration.
- Change the permeabilization protocol, because a high concentration of permeabilization reagent(s) could result in high background.
- Use enough blocking solution such as 5-10% serum to block non-specific staining.
- Include isotype control.

Find additional tips, troubleshooting help, and resources within our Cell Culture Support Center.

Here are some reasons why your cell culture could have failed:

- Did not store cells correctly: These cells should be stored in liquid nitrogen.

- Medium used was not the recommended one: The complete medium of Gibco H9-derived NSCs (Cat. No. N7800) and Gibco StemPro NSCs (Cat. No. A15654 or A15655) have different formulations. Extra components (heparin and ascorbic acid) need to be added into the medium to culture StemPro NSCs.

- Did not thaw cells correctly: Do not thaw the cells for longer than 2 mins at 37 degrees C. After cells have thawed, transfer to 50-mL tube first, then add pre-warmed complete medium drop-wise (approximately 1 drop per second) while swirling the tube.

- Did not count cell viability after thawing cells and did not seed them appropriately: You need to count cell viability with trypan blue. At least 1 x 10E6 viable cells/mL are provided in each vial. For H9-derived NSCs, recommended seeding density is > 1 x 10E5 viable cells/cm2. For StemPro NSCs, recommended seeding density (suspension) is >7 x 10E4 viable cells/mL.

- Plate is not coated or has been coated incorrectly: For H9-derived NSCs and adherent culture, you need to properly coat the tissue culture plate with Gibco Geltrex Matrix, fibronectin, or poly-L-ornithine/laminin, following instructions for coating. For StemPro NSCs, we recommend that you grow them in suspension culture, as adherent culture would trigger differentiation.

Find additional tips, troubleshooting help, and resources within our Cell Culture Support Center.

We offer two types of human NSCs:

- Gibco StemPro Neural Stem Cells (Cat. No. A15654 or A15655): Isolated from human fetal cortex brain and manufactured under good manufacturing practice (GMP). Each lot is generated from the same master bank (same donor) so that the lot-to-lot variability is low. Cell doubling time is ~ 100 hours.

- Gibco Human Neural Stem Cells (H9-derived) (Cat. No. N7800): Induced from H9 human ESC using a proprietary method. Cell doubling time is ~ 40-50 hours and increases with increasing passage number.

Both cells are tested for their ability to retain their proliferation and differentiation potential for at least 3 passsages after thawing. Both are able to differentiate into neurons, astrocytes, and oligodendrocytes. However, StemPro Neural Stem Cells are recommended to grow in suspension culture as adherent culture would trigger differentiation, whereas Human Neural Stem Cells (H9-derived) can grow under both suspension and adherent conditions.

For NSC expansion, the following growth factors are used: recombinant EGF (Cat. No. PHG0314), recombinant FGF-basic (Cat. No. PHG0024), and recombinant VEGF (Cat. No. PHC9394). In addition, several neurotrophins such as BDNF Cat. No. 10908010), NT-3, CNTF (Cat. No. PHC7015), and GDNF (Cat. No. PHC7044)are also used in the related studies.

Information pertaining to whether a specific product has been tested against the WHO Reference Standard can typically be located on the product page or Certificate of Analysis (COA).

Find additional tips, troubleshooting help, and resources within our Cell Culture Support Center.

Human NSCs can grow in Gibco StemPro NSC SFM (Cat. No. A1050901) on dishes pre-coated with Gibco Geltrex Matrix or Gibco CELLstart substrate. Alternatively, if the goal is to obtain neurons, NSCs can also be grown on Neurobasal medium supplemented with Gibco B-27 supplements without vitamin A on a pre-coated dish.

NSCs are generally characterized by their ability to form neurospheres when plated at cloning density (Nat Methods 2:333 (2005)). NSCs can also be characterized by (1) RT-PCR of Sox1, Sox2, and Nestin or (2) immunohistochemical staining for nestin, Pax6, Sox2, and Ki67.

Neural stem cells (NSCs) are self-renewing multipotent cells of the nervous system capable of differentiating into neurons, oligodendrocytes, and astrocytes. NSC can be generated by induced differentiation from embryonic stem (ES) cells, or isolated from various regions of the brain including the cortex, the subventricular zone (SVZ), and the ventricular zone, or generated from bone marrow-derived mesenchymal stem cells (MSCs) (J Cell Biochem 114:764 (2013)). NSCs are valuable tools for the study of neurogenesis and neurotransmitter and receptor function. NSCs were used in the investigation of different CNS disorders such as PD and Huntington's disease in various animal models (J Cell Biochem 114:764 (2013)).