v6.5 Mouse embryonic stem cells

v6.5 Mouse embryonic stem cells Summary

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FAQs for v6.5 Mouse embryonic stem cells (NBP1-41162). (Showing 1 - 10 of 11 FAQs).

1. What passage are the cells at? Has germ line competence been shown for this batch of V6.5 cells?
   • The v6.5 mouse embryonic stem cells are at passage 20. Unfortunately, we have not tested the cells for germline competency.
2. My concerns are as follows: 1. I would like to grow the v6.5 ES cells on mitotically inactivated MEFs, but your protocol indicates that LIF needs to be in the ESL1000 media. Is the 1000 U/ml LIF needed if the cells are growing on mitotically inactivated MEFs (SNL 76/7 or SNLP 76/7-4))? 2. I take it from your answer below that any fetal bovine serum can be used and it does not need to be specified for ES cells or lot tested for ES cells. Is this correct? 3. Can you list for me the critical parameters of the ESL1000 media that are required?
   • 1. Yes, LIF is needed as both feeder layers and exogenously added LIF both serve to prevent differentiation. I only have experience growing the cells with the addition of LIF on top of growing the cells on the MEF feeder layer so I cannot comment on how the cells would grow with the exclusion of LIF. I suppose in theory she could exclude it based on the fact that the MEFs alone can prevent differentiation, however I have not tired that method myself. 2. Again, in my experience I added the FBS that I use for my regular cell culture and did not use one that is specific for ES cells and my cells grew fine, looked healthy and formed nice colonies. However, she may use an ES cell quality FBS if she feels that this is the best approach for her. Just as long as the quality of the FBS is high she should be fine. 3. All ingredients listed in the ESL1000 recipe contained in the protocol are critical to the media. Again, she may substitute out ES cell specific FBS if so desired.
3. What is the passage number of this ESC?
   • The passage number of these ESC is 20.
4. If v6.5 cells are injected into C57mouse blastocysts, are chimeras agouti/black color? How to breed chimeras and test germline transmission?
   • The information we have about chimera color for the v6.5 cell line suggests that the coloring should be agouti. We do not perform experiments on mice here at our facility, we simply bring up and maintain the cell line. For information on how to breed chimeras and test germline transmission, you should consult a colleague or the literature.
5. We are using the V6.5 ES cells, product # NBP1-41162. Have any aliquots from your company been used to create germ-line transmitting chimeras?
   • We are not aware of anyone using NBP1-41162 for this specific use but that does not mean it hasn't been done. We would recommend reading our publications for this antibody to see if they have been used in a similar manner.
6. I am interested in purchasing v6.5 Mouse embryonic stem cells (cat # NBP1-41162). I would like to know whether 2 vials containing 1ml of cells each or a single 2ml vial would be supplied. In addition, I want to know the passage number of the cells that wi
   • Our V6.5 mouse embryonic stem cells are provided as 2.0ml in one vial and the cells are on passage 20.
7. Do you have any chimerism data or myco data for this product?
   • Unfortunately, we do not have any chimeric data for the cells and the cell have not yet been mycoplasma tested.
8. Is the 129/sv of the v6.5 a inbred strain or not, since it's not specifically nomenclated?
   • Yes, 129/sv refers to the inbred strain and you can see more details on the invention of this product in Nature Genetics 24, 109 - 110 (2000). Besides this article, I would encourage you to go through some other publications listed on the datasheet of v65 Mouse embryonic stem cells (NBP1-41162) to see how other researchers have used this product in their experiments.
9. Could you tell us possible passage times of NBP1-41162 after thawing?
   • Exact passage of cells is not known, but the inventor believes they are around passage 20. We routinely test these cells against stem cell markers (SSEA1, Nanog, Oct4, and Sox2) in ICC to confirm that they have not differentiated. Please use our protocol to handle the cells and aliquot/freeze NBP1-41162 right after growing to confluency in T-25. The customer could count 2 x 10*6 cells for one vial to be frozen in liquid nitrogen. After that we think these cells stocks should not be passed over 5-times, as they are already about passage 20. However the number of times NBP1-41162 can really be passed will depend on the the actual experimental condition and media used.
10. What passage number is NBP1-41162?
    • The v6.5 mouse embryonic stem cells are at passage 20. Unfortunately, we have not tested the cells for germline competency. Our V6.5 mouse embryonic stem cells are provided as 2.0ml in one vial.
11. Can you grow the cells feeder free?
    • We do recommend growing on a feeder layer and therefore would not encourage you to grow without MEFs. Here is the link to the protocol specific to these cells: Protocol. This protocol is written for growing cells in T25 tissue culture flasks, please make changes accordingly for flasks of different sizes. ES cells are routinely cultured in ES medium in the presence of LIF on a mitotically inactivated MEF feeder layer grown on gelatin.
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