How do you produce your iPS cells?

From AlleleForum: First off, thank you for choosing Allele Biotech for your iPSC experiment needs. Now onto your questions

You asked Q1: How many human fibroblast cells you normally to start for transfection. I understand you use 12-well plate? How many days you wait till the cells grow confluent? If the cells never grow confluent, should I still transfer them to feeder plate? Is it critical for the cells to reach confluent, if it is, could you suggest the reasons to me

We usually plate at 70% or about 10e4-10e5 cells and transduce the cells for 2-3 days. It should become confluent in 2-3 days. There is no need for the cells to become confluent before splitting onto feeder cells. Please note for primary cells, do not wait for the cells to get too confluent because contact inhibition may induce growth senescence before cells are reprogrammed.

Q2: How many cells you plate on the feeder plate, let’s say it is 6-well plate, and how many clones would normally pup out from each well?

From one well of a 12 well plate, you can plate 1/5 onto a well of a 6 well feeder cell plate. From there, you should get plenty of colonies.

Q3: At the time when you need to cut the Loxp sites, what passage number you do, do you have to dispense the iPS into single cell? Do you have a detailed protocol for that? Other than virus, do you have any other means to do the job, like plasmid?

Never dispense iPSC into single cells. They do not grow back well if split into single cells. iPSC colonies should be passaged in patches of cells. To excise loxP, the suggested timing is after 12-14 days when the cells are reprogrammed into iPSC colonies. Just transduce the iPSC colonies with Cre virus.

Q4: Is it true, that the 4-in-1 is more powerful than individual ones? Do you have the construct(4-in-one) for sale?

The 4-in-1 is somewhat more effective than 4 individual ones. For license issues, we do not distribute the construct to customers because we only offer packaging service. Similar type of plasmid DNAs may be accessible from other sources.

If you have any other questions or concerns, please let us know. Thanks again.

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Wednesday, August 25th, 2010 Allele Mail Bag, iPSCs and other stem cells

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