StemFlex™ Medium - FAQs

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26 product FAQs found

What is the glucose content of the StemFlex Medium (Cat. No. A3349401)?

The StemFlex Medium (Cat. No. A3349401) includes 3.151 g/L (~17.5 mM) of D-glucose (dextrose) in the Basal Medium. The StemFlex Medium Supplement does not include glucose.

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What media do you recommend for standard single-cell cloning of iPSCs?

We recommend StemFlex Medium (Cat. No. A3349401) or Essential 8 (Cat. No. A1517001) for standard single-cell iPSC cloning in the presence of RevitaCell or ROCK inhibitor. StemScale PSC Suspension Medium (Cat. No. A4965001) is not recommended for iPSC cloning.

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Does StemFlex Medium contain pen/strep antibiotic?

StemFlex Medium does not contain pen/strep antibiotic.

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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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Has StemFlex Medium been evaluated for growth of mouse ESCs?

StemFlex Medium contains basic FGF which has been shown to result in differentiation of mouse embryonic stem cells (mESCs) to mouse-derived epiblast stem cells (mEpiSCs). Also, mESCs demonstrate dependence on leukemia inhibitor factor (LIF). Therefore, StemFlex Medium is not recommended for culture of mESCs.

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I am interested in using StemFlex Medium in somatic cell reprogramming. What protocol should I follow for blood cells?

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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I am interested in using StemFlex Medium in somatic cell reprogramming. What protocol should I follow for human fibroblasts?

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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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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Is StemFlex Medium compatible with downstream differentiation kits available from Thermo Fisher Scientific?

Yes. We have seen compatibility with the following differentiation kits provided by Thermo Fisher Scientific: PSC Cardiomyocyte Differentiation Kit (Cat. No. A2921201), PSC Definitive Endoderm Induction Kit (Cat. No. A3062601), PSC Neural Induction Medium (Cat. No. A1647801), and PSC Dopaminergic Neuron Differentiation Kit (Cat. No. A3147701).

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Can I use Geltrex matrix instead of rhLaminin-521 for clonal expansion workflows where cells are seeded at 1-5 cells per well of a 96-well plate?

We have not yet evaluated the Geltrex matrix system for clonal expansion in the presence of StemFlex Medium. However, rhLaminin-521 does provide optimal survival of cells following single-cell passaging and thus this matrix is recommended for such critical applications.

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With StemFlex Medium, what feeding schedule is recommended for seeding of 1-5 cells per well of a 96-well plate for clonal expansion of PSCs?

We recommend feeding of cells every three days, even in the presence of RevitaCell Supplement up to ˜14 days in culture.

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Can antibiotics be used with StemFlex Medium to prevent contamination during gene editing/flow sorting experiments?

Yes. We use Antibiotic-Antimycotic (Cat. No. 15240062) for additional protection from contamination.

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What protocol is recommended for delivery of Cas9 protein:guide RNA complex to my PSCs cultured in StemFlex Medium?

We recommend use of the Neon Electroporation device for electroporation of PSCs with Cas9 protein:guide RNA complex following the protocol guidance in the following demonstrated protocol (https://www.thermofisher.com/content/dam/LifeTech/Documents/PDFs/PG1584-PJT1313-COL31227-Demonstrated-Protocol-Performing-CRISPR-Cas9-FHR.pdf); see the section entitled “Knockout by electroporation of RNP using the Neon Transfection System.” We have seen that the Neon electroporation protocols 7 and 14 provide optimal indel formation while maintaining cell survival. However, the electroporation conditions may need to be optimized for your pluripotent cell line.

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Can PSCs cryopreserved following culture in another feeder-free medium system be thawed directly into the StemFlex Medium system?

Yes. PSCs cryopreserved from cultures of other media systems may be thawed directly into the StemFlex Medium system. However, 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 Versene solution to passage the cells into the StemFlex Medium system.

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I currently use mTeSR1. How difficult would it be to adapt my cells to the StemFlex Medium system?

This transition is very straightforward. We recommend at least a 2-passage transition into the StemFlex Medium system. Briefly, if you have cryopreserved cells previously cultured in the mTeSR1/Matrigel system, we recommend thawing the cells back into mTeSR1/Matrigel until fully recovered. Upon reaching ~70-85% confluency, passage using Versene solution and seed directly into the StemFlex Medium system.

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I see in the flexible feeding schedules specified for StemFlex Medium that cells should be fed the day after passaging, however this is incompatible with my current workflow. Have you evaluated skipping the feed the day after passaging?

Yes. The main reason that the workflow states this is to accommodate ROCK inhibitor or RevitaCell Supplement that have been added during passaging. In such circumstances where ROCK inhibitor or RevitaCell Supplement are added, you should feed your cells within 18-24 hours post-passaging. However, if ROCK inhibitor or RevitaCell Supplement are not included at the time of passaging, then you may skip the day post-passaging.

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I wanted to double-feed the iPSC culture on Friday with StemFlex Medium, but on Saturday, I realized that I forgot about it. What should I do?

In such circumstances, we recommend either full media change on Sunday or overlaying the culture with additional media on Sunday.

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How does StemFlex Medium compare to mTeSR and TeSR-E8?

StemFlex Medium is a more robust medium than TeSR-E8 but has fewer components than mTeSR. Performance of StemFlex Medium was shown to be superior to mTeSR1 in a number of applications including single-cell passaging, gene editing and reprogramming.

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Is the StemFlex Medium xeno-free?

The formulation of StemFlex medium does include BSA and thus is not considered xeno-free. It is more defined than mTeSR1 as it contains less material in the formulation. Essential 8 and Essential 8 Flex are recommended for the most defined and xeno-free PSC culture media.

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You recommend Geltrex as the matrix for routine culture of StemFlex Medium. Can I also use Vitronectin or rhLaminin-521?

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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What should I do if the PSCs are ~100% confluent? Should I discard them or can I continue to passage?

For optimal health of the cultures, cells should be passaged upon reaching ~85% confluency. We have observed improved cell health upon single-cell passaging for cells passaged between 40-85% confluency. If the cells are overly confluent, then inclusion of ROCK inhibitor/RevitaCell Supplement is recommended at the time of passaging. In general, we recommend carrying cells at a range of split ratios to prevent routine passaging at high confluencies as this can result in poor cell survival.

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If I am not interested in the weekend-free feed schedule for StemFlex Medium, can I still feed my cells daily?

StemFlex Medium will accommodate most feeding schedules including every day and weekend-free schedules. For a list of recommended feeding schedules, visit thermofisher.com/stemflex.

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Do I need to use ROCK inhibitor during single-cell passaging in StemFlex Medium?

The use of ROCK inhibitor is not required when culturing in StemFlex Medium on rhLaminin-521. However, supplementation with RevitaCell Supplement can provide additional support to PSCs during stressful transitions such as single-cell passaging.

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What passaging method do you recommend for routine culture of PSCs in StemFlex Medium?

Versene passaging is recommended for routine culture of PSCs in StemFlex Medium.

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I've prepared 1X reconstituted StemFlex Medium aliquots and frozen them at -20 degrees C. Can I thaw this reconstituted medium in a 37 degrees C waterbath?

No. We recommend thawing the aliquot either at room temperature or overnight at 2 to 8 degrees C.

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Can the reconstituted StemFlex Medium be frozen for later use in aliquots?

Yes. Following reconstitution, complete StemFlex Medium can be aliquoted and stored at -5 to -20 degrees C for up to 6 months. Alternatively, aliquots of the supplement can be made and frozen at -5 to -20 degrees C for up to 6 months. Avoid multiple freeze-thaw cycles.

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