Essential 8™ Flex Medium Kit - FAQs

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之前冻存于其他培养条件的多能干细胞（PSCs）能复苏并后续培养于Gibco Essential 8培养基+玻连蛋白（VTN-N）的组合条件中么？

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。

有哪些细胞系在无饲养层培养条件下通过了Gibco Essential 8培养基的测试？

有多株多能干细胞（PSC）系通过了Gibco Essential 8培养基的测试（Chen G, Gulbranson DR, Hou Z et al (2011) Chemically defined conditions for human iPSC derivation and culture.Nat Methods 8:424–429）。

Gibco Essential 8培养基中bFGF的作用是什么？

bFGF对于细胞的存活与增殖至关重要。

Gibco RevitaCell添加剂适用于任何种类的PSC生长培养基么？

我们测试和确认了适用于下列PSC生长培养基：Gibco Essential 8培养基，Gibco StemPro hESC SFM，mTesR1培养基和含有Gibco KnockOut血清替代物（KSR）的饲养层依赖的培养基。

源自皮肤活检样本的成纤维细胞能否培养于Gibco Essential 8培养基中，以实现无异源成份的培养条件？

可以，源自皮肤活检样本的成纤维细胞可在添加有EGF，凝血酶和皮质醇的Gibco Essential 8培养基中培养和增殖（Chen G, Gulbranson DR, Hou Z et al (2010) Chemically defined conditions for human iPSC derivation and culture.Nat Methods 8:424–429.）

Gibco Essential 8培养基中的胰岛素有何作用？

胰岛素对于细胞的存活与增殖至关重要。

细胞可冻存于Gibco Essential 8培养基中么？

是的，细胞可以在完全 Gibco Essential 8 培养基和 10% DMSO 中进行常规冷冻。或者，即用型 CTS PSC Cryomedium（货号 A4238801）可提供更有效的解冻后恢复。

在使用Gibco Essential 8培养基和玻连蛋白（VTN-N）的组合开展细胞培养时，我能否使用分散酶和胶原酶等酶类进行传代？

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。

Gibco Essential 8培养基和Gibco玻连蛋白（VTN-N）的组合中推荐用怎样的方法进行细胞传代？

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。

在Gibco Essential 8培养基中培养细胞时，我能否采用比其他无饲养层培养基更低的换液频率？

不可以。除传代的当天之外，细胞都应每天换液。

Rho相关蛋白激酶（ROCK）抑制剂适用于Gibco Essential 8培养基么？

是的，ROCK 抑制剂可以与 Essential 8 培养基一起使用。我们建议使用 RevitaCell Supplement（货号 A2644501），它专门设计用于最大限度地减少压力对 PSC 的影响。

日常使用时，我能在37°C水浴中温育Gibco Essential 8培养基么？

非常重要的是，Gibco Essential 8完全培养基需放置在室温下进行平衡，请勿在37°C水浴中平衡。bFGF活性会在从4°C至37°C的温度反复改变中迅速降低。

Gibco Essential 8完全培养基的保质期有多久？

Gibco Essential 8完全培养基的保质期为2–8°C条件下放置两周。

我能在37°C水浴中解冻Gibco Essential 8添加剂（50X）么？

我们建议在室温下解冻补充剂约 1 小时。

我能使用其他版本的DMEM/F-12替代试剂盒中所提供的Gibco Essential 8基础培养基么？

在配制Gibco Essential 8培养基时，不能使用其他货号的DMEM/F-12替代Gibco Essential 8基础培养基。试剂盒中提供的Gibco Essential 8基础培养基含有更高浓度的碳酸氢钠。

我应如何配制Gibco Essential 8培养基？

如需配置500 mL 完全Gibco Essential 8培养基，请将Gibco Essential 8添加剂（50X）室温化冻1小时，之后于无菌条件下将下列成份混合在一起：

Gibco Essential 8基础培养基：490 mL
Gibco Essential 8添加剂：10 mL

在Gibco Essential 8培养基 和Gibco玻连蛋白（VTN-N）的体系中培养的细胞外观如何？

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。

之前培养于其他培养基和基质的多能干细胞（PSC）能换用Gibco Essential 8培养基和Gibco玻连蛋白进行培养么？

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。

不同批次的Gibco Essential 8培养基之间会存在差异么？

相比现有其他种类的无饲养层培养基，Gibco Essential 8培养基的变异度更小。不同于其他含有超过20种高度变异性成份的培养基，Gibco Essential 8培养基在cGMP标准下生产，拥有最优化的配方和生长因子水平，能够以最小的变异度帮助用户获得最大的细胞健康度，多能性和生长效果。

Gibco Essential 8培养基与Gibco玻连蛋白（VTN-N）能支持长期培养多能干细胞（PSC）么？

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。。

Gibco Essential 8培养基是不含异源成份（仅人源或重组来源）的么？

是的。Gibco Essential 8培养基不含异源成份，是仅有8种成份的培养基，能够为您提供健康和可靠的培养效果。

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，有哪些主要的区别需加以考虑？

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。

Gibco Essential 8培养基含有bFGF么？

是的。Gibco Essential 8培养基中含有100 ng/mL碱性成纤维细胞生长因子（bFGF），因此无需额外添加bFGF。

Gibco Essential 8培养基中有多少种成份？

Gibco Essential 8培养基仅含培养PSC所需的八种必需成份。本品最初由Chen等（Chen G, Gulbranson DR, Hou Z et al (2010) Chemically defined conditions for human iPSC derivation and culture.Nat Methods 8:424–429).研发，  目的是为了克服mTeSR培养基中所发现的变异度问题。Gibco Essential 8培养基中专门以不含白蛋白（BSA）的有限成份的设计减少变异度。Gibco Essential 8培养基为用户准备了方便的两组分试剂盒：500 mL Gibco Essential 8基础培养基与10 mL Gibco Essential 8添加剂（50X）。

何为Gibco Essential 8培养基？

Gibco Essential 8培养基是一种不含异源成份，无需饲养层的培养基产品，专为人源多能干细胞（PSC）的生长和增殖而配制。Gibco Essential 8培养基最初由James Thomson实验室的Chen等（Chen G, Gulbranson DR, Hou Z et al (2010) Chemically defined conditions for human iPSC derivation and culture.Nat Methods 8:424–429）开发，并通过Cellular Dynamics International验证；到目前为止已久经测试，可维持多类PSC细胞系的多能性。

我能在基因编辑的工作流程中使用Gibco Essential 8 Flex培养基么？

我们目前尚未就基因编辑操作开展Gibco Essential 8 Flex培养基的测试。

我能在重编程研究中使用Gibco Essential 8 Flex培养基么？

我们将本品的使用结果与Gibco Essential 8培养基搭配Invitrogen CytoTune-iPS 2.0 仙台病毒重编程试剂盒的实验结果进行了比较，未发现重编程效率方面受到明显影响。我们推荐在克隆形成阶段每天进行换液（第7-28天）。

你们确认过Gibco Essential 8 Flex培养基培养PSC的三系分化潜能么？

对于Gibco Essential 8 Flex培养基中长期培养的细胞，其分化为外胚层，内胚层，中胚层谱系的潜力不受影响。这一结论是由拟胚体的自发分化实验和神经干细胞，心肌细胞和定形内胚层的定向分化实验确认过的。

你们是否在Gibco Essential 8 Flex培养基培养的PSC中观察到多能标志物表达的某些改变？

我们在Gibco Essential 8 Flex培养基培养多株PSC细胞系超过50代，未观察到多能标志物表达方面的显著改变。

我是否会在Gibco Essential 8培养基与Gibco Essential 8 Flex培养基中培养的两批细胞之间观察到形态差异？

培养在Gibco Essential 8 Flex培养基中的细胞能够保持PSC的正常形态，如紧凑均一的细胞克隆，清晰边界和高核质比等特征。

我能否将Gibco Essential 8 Flex培养基中长期培养的PSC用于后续的分化操作？

我们将细胞培养在Gibco Essential 8 Flex培养基中长达15代，未发现细胞的后续分化潜能受到任何影响。进行拟胚体分化和使用Gibco PSC神经诱导培养基（货号A1647801），Gibco PSC心肌细胞分化试剂盒（货号A25042SA），Gibco PSC定形内胚层诱导试剂盒（货号A27654SA）等产品进行分化均证明这些细胞拥有三系分化潜能。

在Gibco Essential 8 Flex培养基中常规培养细胞是否会对我的细胞的核型造成影响？

我们发现长期培养长达50代的多株PSC细胞系具有正常的核型。

使用Gibco Essential 8 Flex培养基时，我的细胞在两天不换液间隔期中是否会累积废物而产生不良效应？

和任何PSC培养一样，使用Gibco Essential 8 Flex培养基可能会出现一些细胞碎片和pH值的下降情况。不过，我们未发现这些累积的废物对细胞生长或多能性方面造成长期的不良影响。

我的细胞适应了Gibco Essential 8 Flex培养基之后，稳定的换液时间表和连续的无饲养层培养会对这些细胞有何长期影响？

我们在省略每天换液的条件下，使用Gibco Essential 8 Flex培养基培养多种细胞系多达50代，未发现明显影响。多能性标志物的表达（Tra-1-60，SSEA4，Sox2，Oct-4与Nanog），分化潜能与正常核型在Gibco Essential 8 Flex培养基长期培养的条件下均得到保持。

StemBeads FGF2添加剂的工作流程与Gibco Essential 8 Flex培养基的相比有何异同？

不同于StemBeads FGF2添加剂，Gibco Essential 8 Flex培养基无需任何额外添加或优化操作，即可有效限制FGF2的活性丧失。

Gibco Essential 8 Flex培养基与缓释FGF2添加剂之间的区别是什么？

StemBeads FGF2添加剂一类的缓释产品能够向生长培养基中释放FGF2，从而补充随时间而逐渐丢失的FGF2活性，而Gibco Essential 8 Flex培养基则能够避免FGF2的活性丧失。

我正在使用一款现有的无饲养层培养基，不过希望能免去周末的换液操作。我能仅在周末换用Gibco Essential 8 Flex培养基么？

由于持续使用单一的PSC培养基能够帮助获得最佳的培养效果，我们不推荐频繁地将细胞从一种培养基直接切换至另一种。持续使用Gibco Essential 8 Flex培养基能够获得最佳结果。

我目前正在使用Gibco Essential 8培养基，我能方便地切换至Gibco Essential 8 Flex培养基么？我能仅在周末离开时使用这款培养基么？

持续使用同款培养基培养您的PSC细胞将获得最佳结果。从Gibco Essential 8培养基切换至Gibco Essential 8 Flex培养基是一个十分简单的操作过程，您只需在一周开始时将细胞接种到Gibco Essential 8 Flex培养基中即可，在进入两天不换液的间隔期之前，最好对细胞进行一次传代操作。

我能否对培养在Gibco Essential 8 Flex培养基中的细胞进行冻存？

可以，在Gibco Essential 8 Flex培养基中常规培养的细胞可进行冻存和复苏，相关操作与使用Gibco Essential 8培养基一致。

在使用Gibco Essential 8 Flex培养基的条件下，我能否频繁调整换液时间表？

请选择并保持规律的传代和换液操作时间以得到最优的结果。举例来说，如果您希望周末不换液，您可在周一/周四分种细胞，周二/周五进行换液。

我能否使用Gibco Essential 8 Flex培养基进行单细胞传代？

可以，Gibco Essential 8 Flex培养基能够兼容单细胞传代操作。当您进行单细胞传代时，请确保使用了Gibco RevitaCell添加剂（货号 A2644501）或一些其他种类的ROCK抑制剂。

能否在Gibco Essential 8 Flex培养基中使用ROCK抑制剂？

可以，Gibco Essential 8 Flex培养基中可添加ROCK抑制剂。我们推荐您使用Gibco RevitaCell添加剂（货号 A2644501），该产品专为最大程度地减少应激对PSC的影响而设计。

日常使用时，我能在37°C水浴中温育Gibco Essential 8 Flex培养基么？

不可以，请勿在37°C条件下温育Gibco Essential 8 Flex培养基。将其缓慢平衡至室温效果最佳。

加入添加剂后的Gibco Essential 8 Flex培养基保质期有多久？

为达到最佳效果，加入添加剂后的Gibco Essential 8 Flex完全培养基建议两周内用完。

在Gibco Essential 8 Flex添加剂的化冻过程中，我观察到一些沉淀，我该怎么办？

Gibco Essential 8 Flex添加剂中出现沉淀现象是比较少见的；不过，即使发现有沉淀，也不会影响培养基的使用效果。如果您在添加剂中观察到沉淀，请充分混匀后按常规操作将其加入Gibco Essential 8 Flex基础培养基中。  

我能在37°C水浴中化冻Gibco Essential 8 Flex添加剂么？

不可以，Gibco Essential 8 Flex添加剂不宜在37°C水浴中化冻。在室温条件下化冻1小时左右可获得最佳结果。您也可在2–8°C条件下过夜化冻Gibco Essential 8 Flex添加剂，这样的操作可能会产生少量沉淀物。沉淀物的存在不会对培养基使用效果产生负面影响。

我可以在Gibco Essential 8基础培养基中加入Gibco Essential 8 Flex添加剂么？

不可以，Gibco Essential 8 Flex培养基是一个完整的试剂盒，其中基础培养基与冻存添加剂需配套使用。Gibco Essential 8 Flex培养基的组份不应与Gibco Essential 8培养基的组份混合。

若尝试从Gibco Essential 8原始培养基切换至Gibco Essential 8 Flex培养基，在配制完全培养基的方法上有什么不同？

Gibco Essential 8 Flex培养基的配制方法与Gibco Essential 8 原始培养基完全相同。唯一的差别在于换液的时间安排，Gibco Essential 8 Flex培养基所需的换液间隔时间更为灵活，可维持连续两天无需换液。  

使用Gibco Essential 8 Flex培养基进行换液和传代的时间应怎样安排？

我们在每周两次传代细胞，铺板24小时左右进行换液的条件下观察到了最优结果。周一和周四进行传代，周二和周五（双倍培养基体积）换液结果最佳。

Gibco Essential 8 Flex培养基的推荐传代比例是多少？

传代比例需基于您所使用的细胞系来确定。Gibco Essential 8 Flex培养基通常能够良好兼容1:3至1:12的分种比例。

你们仍推荐使用Gibco玻连蛋白作为Gibco Essential 8 Flex培养基的包被基质么？

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。  

Gibco Essential 8 Flex培养基与其他无需周末换液的PSC培养溶液有何区别？

不同于其他免除了周末换液操作的市售商品化PSC培养液，Gibco Essential 8 Flex培养基不需要为了避免换液而调节传代细胞的比例。有了Gibco Essential 8 Flex培养基，您就可维持一周两次的分种安排并省去周末的换液操作了。  

我应何时开始使用Gibco Essential 8 Flex培养基进行换液？

在每周初换用Gibco Essential 8 Flex培养基可观察到最佳效果，Gibco Essential 8 Flex培养基常规用于已建立好的无饲养层培养系统。

Gibco Essential 8 Flex培养基的换液时间安排有多灵活？你们能例举出一份换液时间表吗？

在每周两次传代细胞的频率下可观察到最佳结果。此处以一份无需周末换液的培养时间表为例，用户可在周一和周四分种细胞，并在传代后24小时左右——即周二和周五进行一次培养基的彻底更换。在该例中，周五换液添加的培养基应是标准体积的两倍。  

使用Gibco Essential 8 Flex培养基时，我每周几天无需换液？

您最多可连续两天不换液（如周六周日），每周最多三天不换液。

Gibco Essential 8 Flex培养基的产品设定与Gibco Essential 8 原始培养基有所不同么？

不是的，Gibco Essential 8 Flex培养基与Gibco Essential 8 培养基的产品设定是相同的；也是基础培养基与冻存的50X添加剂一并提供。

Gibco Essential 8 Flex培养基是如何由Gibco Essential 8培养基的原始配方调整而来，以适应用户更为灵活的培养时间安排？

成份明确的Gibco Essential 8原始培养基配方经专门优化，能够延长PSC培养时两次换液的间隔时间。

Gibco Essential 8 Flex培养基如何实现无需每日换液？

Gibco Essential 8 Flex培养基是在Gibco Essential 8原始培养基的基础上通过配方调整而得来，延长了PSC培养基中关键热敏成份（包括FGF2）的活性。

What is the pH range for Essential 8 Medium?

The pH range for Essential 8 Medium is pH 7 -7.4.

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Where can I find the osmolality for Essential 8 Medium?

We do provide osmolality information on the certificate of analysis for the particular lot number of your medium. All lots of Essential 8 Medium will meet the osmolality specification of ≥290 to ≤340 mOsm/kg.

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Can I filter sterilize Essential 8 Medium using a 0.22 µm filter?

You can filter the basal Essential 8 medium or the complete Essential 8 medium through a 0.22 µm filter (vacuum driving filtration). But, we recommend that you use a low protein-binding filter.

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What is the difference between StemFlex Medium and Essential 8 Flex Medium?

Essential 8 Flex Medium is a serum-free, xeno-free medium that contains proprietary material to extend the activity of the key heat-sensitive component, FGF2. Therefore, it supports the culture and expansion of feeder-free pluripotent stem cells (PSCs) without the need for daily or weekend feeding. Note: We also offer Essential 8 Medium that does not allow skipping of PSC feeding during the weekend.

StemFlex Medium also supports the expansion of feeder-free PSCs with a flexible feeding schedule (including weekend-free feeding) and also the ability to choose the matrix and passaging reagent that best suits specific applications.

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Is it possible to reprogram somatic cells using Essential 7 or Essential 8 medium and transition them into StemFlex Medium upon harvest?

Yes. If improved reprogramming efficiency is required, then you may utilize Essential 7 (Essential 6 + bFGF) or Essential 8 Medium for reprogramming somatic cells and directly transition them into StemFlex Medium system upon colony selection.

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Can pluripotent stem cells (PSCs) cryopreserved in a different culture condition be thawed and subsequently cultured in Gibco Essential 8 Medium and on vitronectin (VTN-N)?

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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What cell lines have been successfully tested with Gibco Essential 8 Medium under feeder-free conditions?

There have been multiple pluripotent stem cell (PSC) lines tested with the Gibco Essential 8 Medium System (https://www.ncbi.nlm.nih.gov/pubmed/?term=21478862).

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What is the role of bFGF in Gibco Essential 8 Medium?

bFGF is vital for pluripotent cell survival and proliferation.

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Can Gibco RevitaCell Supplement be used with any PSC growth medium?

We have tested and confirmed utility with the following PSC growth media: Gibco Essential 8 Medium, Gibco StemPro hESC SFM, mTesR1 medium, and Gibco KnockOut Serum Replacement (KSR)- containing feeder-dependent medium.

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Can fibroblasts obtained from skin biopsy samples be cultured in Gibco Essential 8 Medium to achieve xeno-free conditions?

Yes, fibroblasts from skin biopsy samples can be expanded and cultured in Gibco Essential 8 Medium with the addition of EGF, thrombin, and hydrocortisone (http://www.ncbi.nlm.nih.gov/pubmed/21478862).

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What is the role of insulin in Gibco Essential 8 Medium?

Insulin is important for cell survival and proliferation.

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Can human PSCs be frozen in Gibco Essential 8 Medium?

Yes, cells can be routinely frozen in complete Gibco Essential 8 Medium and 10% DMSO. Alternatively, ready-to-use CTS PSC Cryomedium (Cat. No. A4238801) provides more efficient post-thaw recovery.

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Can I use enzymes such as dispase and collagenase for passaging cells cultured in Gibco Essential 8 Medium and on VTN-N?

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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What is the recommended passaging method to use with Gibco Essential 8 Medium and on Gibco Vitronectin (VTN-N)?

Cells cultured in Gibco Essential 8 Medium and VTN-N need to be passaged with EDTA.

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Can I feed the cells grown in Gibco Essential 8 Medium less frequently than those in other feeder-free medium?

No. The cells should be fed daily including the day after passaging.

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Can Rho-associated protein kinase (ROCK) inhibitors be used in Gibco Essential 8 Medium?

Yes, ROCK inhibitors can be used with Essential 8 Medium. We suggest using RevitaCell Supplement (Cat. No. A2644501), which has been specifically designed to minimize the impact of stress on PSCs.

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Can I warm Gibco Essential 8 Medium in a 37 degrees C waterbath for daily use?

It is very important that complete Gibco Essential 8 Medium is prewarmed at room temperature and not in a 37 degrees C water bath. bFGF activity can decline rapidly with repeated temperature changes from 4 degrees C to 37 degrees C.

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What is the shelf life of complete Gibco Essential 8 Medium?

The shelf life of complete Gibco Essential 8 Medium is two weeks at 2-8 degrees C.

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Can I thaw the frozen Gibco Essential 8 Supplement (50X) in a 37 degrees C waterbath?

We recommend thawing the supplement at room temperature for approximately 1 hour.

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Can I use other versions of DMEM/F-12 in place of the Gibco Essential 8 Basal Medium supplied with the kit?

Other catalog versions of DMEM/F-12 cannot be used in place of the Gibco Essential 8 Basal Medium in the preparation of Gibco Essential 8 Medium. Gibco Essential 8 Basal Medium supplied with the kit has a higher level of sodium bicarbonate.

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How do I prepare Gibco Essential 8 Medium?

To prepare 500 mL of complete Gibco Essential 8 Medium, thaw Gibco Essential 8 Supplement (50X) at room temperature for 1 hour and then aseptically combine the components listed below:

- Gibco Essential 8 Basal Medium: 490 mL
- Gibco Essential 8 Supplement (50X): 10 mL

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What will my cells look like growing in Gibco Essential 8 Medium and on Gibco Vitronectin (VTN-N)?

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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Can pluripotent stem cells (PSCs) previously cultured in other media and on other substrates be cultured in Gibco Essential 8 Medium and on Gibco Vitronectin?

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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Does Gibco Essential 8 Medium exhibit variability from lot to lot?

Gibco Essential 8 Medium has reduced variability compared to existing feeder-free culture media. Unlike other media that contain over 20 highly variable ingredients, Gibco Essential 8 Medium is produced under cGMP and has an optimized formulation and growth factor levels that help ensure maximum cell health, pluripotency, and growth, with minimal variability.

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Can Gibco Essential 8 Medium and Gibco Vitronectin (VTN-N) support long-term growth of pluripotent stem cells (PSCs)?

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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Is Gibco Essential 8 Medium xeno-free (human or recombinant origin only)?

Yes. Gibco Essential 8 Medium provides reliable and robust cultures with a xeno-free, eight-component medium

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What are the main 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?

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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Does Gibco Essential 8 Medium already contain bFGF?

Yes. Gibco Essential 8 Medium contains 100 ng/mL basic fibroblast growth factor (bFGF), and no additional bFGF is required.

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How many components are there in Gibco Essential 8 Medium?

Gibco Essential 8 Medium contains only the eight required components for culturing PSCs. The medium was developed by Chen et al (http://www.ncbi.nlm.nih.gov/pubmed/21478862) to overcome the variability issues observed with mTeSR Medium. Gibco Essential 8 Medium is designed to have less variability due to limited components and removal of albumin (BSA) from the formulation. Gibco Essential 8 Medium is provided as a convenient two-component kit: 500 mL Gibco Essential 8 Basal Medium and 10 mL Gibco Essential 8 Supplement (50X).

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What is Gibco Essential 8 Medium?

Gibco Essential 8 Medium is a xeno-free and feeder-free medium specially formulated for the growth and expansion of human pluripotent stem cells (PSCs). Originally developed by Chen et al (http://www.ncbi.nlm.nih.gov/pubmed/21478862) in the laboratory of James Thomson, and validated by Cellular Dynamics International, Gibco Essential 8 Medium has been extensively tested and is proven to maintain pluripotency in multiple PSC lines.

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Can PSCs transitioned over into Essential 8 Medium on recombinant human laminin-521 (rhLaminin-521) from feeder-dependent cultures, then be transferred onto vitronectin (VTN-N)?

Yes. Following 2 passages on the rhLaminin-521 matrix, Versene or EDTA passaging should be used to subculture PSCs.

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Can recombinant human laminin-521 (rhLaminin-521) be used with Essential 8 Flex Medium?

Yes. To ensure optimum recovery of PSCs following single-cell passaging, PSCs should be fed with Essential 8 Flex Medium the day before passaging.

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Can I use Gibco Essential 8 Flex Medium in my gene editing workflow?

We have not tested Essential 8 Flex Medium with gene editing at this time.

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Can I use Gibco Essential 8 Flex Medium in my reprogramming studies?

We have seen no effect on reprogramming efficiency as compared to Gibco Essential 8 Medium with Invitrogen CytoTune -iPS 2.0 Sendai Reprogramming Kit. We do recommend daily feeding during colony formation (days 7 - 28).

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Have you confirmed tri-lineage differentiation potential from PSCs cultured in Gibco Essential 8 Flex Medium?

The potential for cells to differentiate into cells of ectoderm, endoderm and mesoderm lineages is unaffected by long term culture in Essential 8 Flex Medium. This has been confirmed using both spontaneous differentiation from embryoid bodies and directed differentiation to neural stem cells, cardiomyocytes and definitive endoderm.

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Do you see any change in pluripotency marker expression from PSCs cultured in Gibco Essential 8 Flex Medium?

We have observed no significant change in pluripotency marker expression in multiple cell lines over 50 passages in Essential 8 Flex Medium.

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Will I see a morphological difference between cells cultured in Gibco Essential 8 Medium and cells cultured in Gibco Essential 8 Flex Medium?

Cells cultured in Essential 8 Flex Medium maintain the expected PSC morphology with compact homogeneous colonies, defined edges and a high nucleus-to-cytoplasm ratio.

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Can I use PSCs that are long-term cultured in Gibco Essential 8 Flex Medium for downstream differentiation?

We have seen no effect on downstream differentiation potential for cells cultured in Essential 8 Flex Medium for up to 15 passages. Tri-lineage potential has been demonstrated from embryoid bodies as well as using Gibco PSC Neural Induction Medium (Cat. No. A1647801), Gibco PSC Cardiomyocyte Differentiation Kit (Cat. No. A25042SA) and Gibco PSC Definitive Endoderm Induction Kit (Cat. No. A27654SA).

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Does routine culture in Gibco Essential 8 Flex Medium have any effect on the karyotype of my cells?

We have observed normal karyotypes in multiple PSC lines with long-term culture up to 50 passages.

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Is there an effect of accumulated waste product build up during my two-day feed-free period when using Gibco Essential 8 Flex Medium?

As with any PSC culture, some cell debris and pH drift will occur with Essential 8 Flex Medium. However, we have observed no adverse long-term effects from waste product buildup on either cell growth or pluripotency.

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What is the long-term impact on my cultures after adopting Gibco Essential 8 Flex Medium with a consistent feed schedule with consecutive feeder-free days?

We have observed no impact from skipping daily feeding with Essential 8 Flex Medium for up to 50 passages in multiple cell lines. Pluripotency marker expression (Tra-1-60, SSEA4, Sox2, Oct-4, and Nanog), differentiation potential and normal karyotypes are all maintained in long-term culture with Essential 8 Flex Medium.

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How does the workflow for StemBeads FGF2 compare to the workflow for Gibco Essential 8 Flex Medium?

Unlike StemBeads FGF2, Essential 8 Flex Medium does not require any additional supplementation or optimization to limit the loss of FGF2 activity.

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What is the difference between Gibco Essential 8 Flex Medium and a time-release FGF2 substitute?

Time-release products such as StemBeads FGF2 release FGF2 into the growth medium to counteract the loss of FGF2 activity over time, while Essential 8 Flex Medium prevents the loss of FGF2 activity from even happening.

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I already use an existing feeder-free medium but would love to take a weekend off from maintaining my cultures. Can I switch to Gibco Essential 8 Flex Medium for just a weekend?

Because optimal PSC culture results are achieved using a single PSC culture medium, we do not recommend routinely switching cells from one medium to another. Optimal results are achieved when Essential 8 Flex Medium is used and maintained as the primary culture system.

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If I am currently using Gibco Essential 8 Medium, can I easily transition to Gibco Essential 8 Flex Medium? Can I use it for just a weekend if I am going to be away?

Best results are observed when you maintain your PSCs consistently in one medium. Transitioning from Essential 8 Medium to Essential 8 Flex Medium is as simple as seeding your cells into Essential 8 Flex Medium at the beginning of the week. Best results are achieved if the cells are split one time prior to the 2-day feed-free period.

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Can I cryopreserve cells cultured in Gibco Essential 8 Flex Medium?

Yes, cells that are routinely cultured in Essential 8 Flex Medium can be cryopreserved and revived just as they are in Gibco Essential 8 Medium

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Can I frequently change the feeding schedule with Gibco Essential 8 Flex Medium?

Best results are observed if you choose and maintain a regular split and feed schedule. For example, if you desire a weekend-free feeding schedule, you should split Monday/Thursday and feed on Tuesday/Friday.

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Can I perform single cell passaging with Gibco Essential 8 Flex Medium?

Yes, Essential 8 Flex Medium is compatible with single cell passaging. Whenever you are performing single cell passaging, ensure that you use Gibco RevitaCell Supplement (Cat. No. A2644501) or another ROCK inhibitor.

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Can I use ROCK inhibitors with Gibco Essential 8 Flex Medium?

Yes, Essential 8 Flex Medium has been used with ROCK inhibitors. We suggest using Gibco RevitaCell Supplement (Cat. No. A2644501), which has been specifically designed to minimize the impact of stress on PSCs.

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Can I warm Gibco Essential 8 Flex Medium in a 37 degrees C water bath for daily use?

No, do not warm the Essential 8 Flex Medium at 37°C. Best results are achieved when the medium is allowed to slowly reach room temperature.

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What is the shelf life of Gibco Essential 8 Flex Medium after supplementation?

The best results are observed when complete Essential 8 Flex Medium is used within 2 weeks of supplementation.

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If I see precipitation when I thaw the Gibco Essential 8 Flex Supplement, what should I do?

The appearance of precipitate in the Essential 8 Flex Supplement is rare; however, if it is seen, it should not affect the performance of the medium. If you see precipitation in the supplement, keep it well mixed and add it to the Gibco Essential 8 Flex Basal Medium as you normally would.

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Can I thaw the Gibco Essential 8 Flex Supplement in a 37 degrees C water bath?

No, do not thaw the Essential 8 Flex Supplement in a 37°C water bath. Best results are observed thawing at room temperature for approximately 1 hour.

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Can I use Gibco Essential 8 Base Medium with Gibco Essential 8 Flex Supplement?

No, Gibco Essential 8 Flex Medium is a complete kit, with a base medium and frozen supplement designed to be used together. Gibco Essential 8 Flex Medium components should not be mixed with Gibco Essential 8 Medium components.

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If I intend to transition from the original Gibco Essential 8 Medium to Gibco Essential 8 Flex Medium, will I need to prepare the complete medium any differently?

The preparation of Essential 8 Flex Medium is equivalent to that of the original Essential 8 Medium. The only difference is the feeding schedule, with Essential 8 Flex Medium enabling a more flexible, 2-day break between required culture feeds.

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What is the timing for medium changes and passaging when using Gibco Essential 8 Flex Medium?

Optimal results are observed when the cells are split twice weekly with a medium exchange approximately 24 hours after plating. Splitting Monday and Thursday with Tuesday and Friday (double volume) feeds have shown the best results.

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What are the recommended passaging ratios when using Gibco Essential 8 Flex Medium?

Passaging ratios depend on the cell line you are using. Split ratios between 1:3 and 1:12 are generally well tolerated in Essential 8 Flex Medium.

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Do I need to add a ROCK inhibitor during passaging when culturing in Gibco Essential 8 Flex Medium?

As with many other PSC media, ROCK inhibitor is not required with Essential 8 Flex Medium when Gibco Versene Solution is used to passage PSCs as clusters. If cells are plated as singlets, we suggest that Gibco RevitaCell Supplement (Cat. No A2644501) or another ROCK inhibitor be used.

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What other dissociation reagents have been tested with Gibco Essential 8 Flex Medium?

Gibco TrypLE Select Enzyme (Cat. No. 12563011) and Gibco StemPro Accutase Cell Dissociation Reagent (Cat. No A1110501) have been tested with Essential 8 Flex Medium.

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Is Gibco Versene Solution still the recommended dissociation reagent with Gibco Essential 8 Flex Medium?

Both Versene Solution (Cat. No. 15040066) and enzyme-mediated dissociation have been shown to be compatible passaging methods.

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Do you still recommend using Gibco Vitronectin as the matrix for Gibco Essential 8 Flex Medium?

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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How does Gibco Essential 8 Flex Medium differ from other weekend-free PSC culture solutions?

Unlike other commercially available PSC culture solutions that also offer weekend-free feeding, Essential 8 Flex Medium does not require substantial split ratio adjustments prior to the break in feeding. With Essential 8 Flex Medium, you can maintain your twice-weekly split schedule and skip weekend feeds.

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When should I start feeding my cells with Gibco Essential 8 Flex Medium?

Best results are observed when cultures are switched into Essential 8 Flex Medium at the start of the week and Essential 8 Flex Medium is routinely used in place of existing feeder-free culture systems.

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How flexible is the Gibco Essential 8 Flex Medium feeding schedule? Can you give an example feeding schedule?

Best results are observed when cells are split twice weekly. To enable a weekend-free culture schedule, cells would be split on Monday and Thursday with full media exchanges on Tuesday and Friday, approximately 24 hours after plating. In this example, the Friday feed should use twice the standard volume.

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How many days a week can I skip feeding my culture when using Gibco Essential 8 Flex Medium?

You are able to skip feeding up to two consecutive days (e.g., Saturday and Sunday) and up to three days per week.

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Is the product configuration of Gibco Essential 8 Flex Medium different from the original Gibco Essential 8 Medium?

No, the Essential 8 Flex Medium product configuration is the same as that of Essential 8 Medium; a basal medium with a 50X Supplement.

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How has the original Gibco Essential 8 Medium formulation been modified to enable a more flexible culture schedule with Gibco Essential 8 Flex Medium?

The original, defined Essential 8 Medium formulation has been optimized to allow for an extended period of time between PSC culture feedings.

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How does Gibco Essential 8 Flex Medium work without daily feeding?

Essential 8 Flex Medium has been reformulated from the original Gibco Essential 8 Medium to extend the activity of key heat-sensitive compounents found in PSC medium, including FGF2.

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