CytoTune™ EmGFP Sendai Fluorescence Reporter - FAQs

如果您希望在重编程过程中应用EmGFP报告基因，就必须在重编程操作的同时加入。感染过仙台病毒的细胞对于后续的感染会产生抵抗。因此，不要试图对已经转导了Invitrogen CytoTune-EmGFP仙台病毒荧光报告基因的细胞加入Invitrogen CytoTune-iPS 2.0仙台病毒重编程试剂，反之亦然。

成功转导细胞中的EmGFP的表达在转导后的24小时可通过荧光显微镜检测到，在转导后的48-72小时会达到最高水平。

将报告基因一次性加入细胞，再监测其表达情况。不同的细胞类型接纳仙台病毒的能力有所不同；因此，我们推荐用户在开始阶段尝试使用2-3种不同的MOI值（如1,3,9）转导您的细胞。请参考用户手册（https://tools.thermofisher.com/content/sfs/manuals/cytotune_emgfp_sendai_reporter_man.pdf）获取完整的实验方案。

祖母绿荧光蛋白（EmGFP）是一种更为明亮的GFP蛋白，可用于监测目的基因的表达情况。EmGFP可通过标准的FITC通道进行检测。  

Invitrogen CytoTune-EmGFP仙台病毒荧光报告基因是一个携带EmGFP基因的荧光对照载体。荧光对照载体能够帮助用户确定所关注的细胞能够接受还是抵抗仙台病毒重编程载体的感染。  

人iPS与人ES细胞的生长条件相同。与人ES细胞类似，人iPS细胞能够生长于辐照的MEF滋养层细胞上+含20% KnockOut SR，0.1 mM NEAA，1mM谷氨酰胺，0.1mM b-ME的DMEM/F12培养基 + 辐照MEF饲养细胞 + 100 ng/mL bFGF的培养条件中，或Matrigel基质包被培养皿 + MEF条件培养基 + 100 ng/mL bFGF的培养条件中（Science318:1917 (2007)）。人ES细胞与iPS细胞两者均可生长于KnockOut ESC/iPS培养基中（货号A14131）。

用于细胞培养：人重组激活化素A（货号PHC9564），bFGF（货号PHG0261），IGF-II（货号PHG0084）（Stem Cells 24:1476 (2006); Nature 448:1015 (2007)）。
用于人ES细胞分化：BMP-4（货号PHC9533），EGF（货号PHG0311）和HGF（货号PHG0324）（Proc Natl Acad Sci U S A 97:11307 (2000)）。

If you want to use the EmGFP Reporter with reprogramming, it must be added at the time of reprogramming. Cells infected with Sendai virus will most likely be refractive to further infection. Therefore, do not try to add Invitrogen CytoTune-iPS 2.0 Sendai Reprogramming Kit to cells already transduced with Invitrogen CytoTune-EmGFP Sendai Fluorescence Reporter or vice versa.

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

The expression of EmGFP in successfully transduced cells is detectable at 24 hours post-transduction by fluorescence microscopy and reaches maximal levels at 48-72 hours post-transduction.

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

Add the reporter one time to your cells and monitor for expression. Different cell types may vary in their ability to take up Sendai virus; therefore, we suggest initially transducing your cells with at least 2-3 different MOIs (e.g., 1, 3, and 9). Please refer to the user manual for the full protocol (http://tools.thermofisher.com/content/sfs/manuals/cytotune_emgfp_sendai_reporter_man.pdf).

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

Emerald Green Fluorescent Protein (EmGFP) is a form of GFP with brighter green fluorescence, used to report the expression of a gene of interest. EmGFP can be visualized on standard FITC channel.

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

The Invitrogen CytoTune-EmGFP Sendai Fluorescence Reporter is a fluorescent control vector carrying the EmGFP gene. The fluorescent control vector allows the determination of whether a cell line of interest is amenable or refractive to infection by Sendai reprogramming vectors.

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

The full length Human FGF-basic (FGF-2/bFGF) (aa 1-155) Recombinant Protein is recommended for stem cells whereas the truncated variant, Human FGF-basic (FGF-2/bFGF) (aa 10-155) Recombinant Protein which is missing the first 9 amino acids, is recommended for use with neural and cardiac cells.

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

Induced pluripotent stem cells (iPS or iPSCs) are pluripotent stem cells directly generated by introducing combination of genes coding for “reprogramming factors” into adult cells. These reprogramming factors include Oct4, Sox2, c-Myc, KLF4, NANOG, and LIN28. Yu, et al, generated iPS from a human mesenchymal cell line using lentiviral vectors carrying Oct4, Sox2, NANOG, and LIN28 genes (Science 318:1917 (2007)). Using a similar approach, Takahashi et al, generated iPS from human primary fibroblast cells by introducing genes coding for Oct3, Sox2, KLF4, and c-Myc into these cells (Cell 131:861 (2007)). iPS generated by reprogramming are similar to human ES cells in morphology, the capacity for unlimited proliferation, surface-antigen expression, gene expression, the ability to differentiate into cell types representing the three germ layers in vitro, and the ability to form teratomas after injection into SCID mice.