rhLaminin-521, 100μg - FAQs

View additional product information for rhLaminin-521 - FAQs (A29248, A29249)

14 product FAQs found

What is the alternative for rhLaminin-521 (Cat. No. A29249)?

The appropriate alternative for rhLaminin-521 (Cat. No. A29249) would be either Laminin Mouse Protein, Natural (Cat. No. 23017015) or Vitronectin (VTN-N) Recombinant Human Protein, Truncated (Cat. No. A14700).

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

Can I use the Poly-D-Lysine matrix with other coatings?

iPSC-derived neurons have been cultured successfully on Poly-D-Lysine with a secondary coating of mouse and human laminin.

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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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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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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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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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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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Can I use EDTA or versene for passaging of stem cells?

Yes, you may use EDTA or versene. However, we do not recommend using dispase or collagenase as it can lead to differentiation.

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Does the recombinant human laminin-521 (rhLaminin-521) have to be on ice before use?

We recommend that you leave the product on ice while prepping to use it.

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Do recombinant human laminin-521 (rhLaminin-521) pre-coated plates have to be covered with liquid at all times?

Yes, do not let the laminin dry out on the plates.

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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 at coating concentrations lower than 0.5 ug/cm2?

The optimal working concentration of rhLaminin-521 is cell line dependent and must be determined empirically. However, for some cell lines, coating concentrations as low as 0.3 µg/cm2 can be used with no decrease in performance. Additionally, coating plates overnight at 4 degrees C can support coating concentrations as low as 0.1 µg/cm2.

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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.

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