Which concentration of Matrigel you use for iPSC culture? I was using 500 ul in 15ml and then, I have added 2 ml in 6 cm dish. However, my colonies did not attach.
Now, I am using 1.5ml of matrigel in 12 ml and so far, nothing.
:)
In my case I use Corning Matrigel and you have to use the dilution factor that appears in the "Certificate of analysis" page of your product. In this page you can see which is the dilution factor that you have to use. The number that appears there is the amount that you have to use for 25 ml of medium. Then you can calculate what you need for your plates.
Using this dilution factor I've never had had problems relating to cell atachment.
Good luck!
Yes, I have read something it.
But I am still have issue to understand this dilution factor.
Could you give me an example of preparation of matrigel?
In the lot I'm using now the Dilution Factor is 260. This means that I've to dilute 260 ul in 25 ml of cold medium. Normally I work with p60 plates so for one plate I dilute 31 ul of matrigel in 3 ml of medium, which is the amount that I use for a p60 plate.
ok, I understood.
DO you use the corning matrigel hESC-qualified matrix ( Cat no. 354277) ?
Hi,
I use a 1:30 dilution. iPS cells do not attach when: 1) iPS cells are differentiated or death 2) Matrigel is too old, 3) Matrigel is dry, 4) Medium is too old, 5) iPSCs are cells are improperly passaged.
Using a dilution ratio is meaningless unless you know the actual concentration of the matrigel you have on hand. I am not aware of any vendor that supplies it at a constant concentration that would allow you to use a constant dilution ratio.
All that matters is the protein concentration of the specific lot of matrigel that you have. From that you can calculate the proper dilution. In general, I use one mg of matrigel per 100 mm plate, which is the same surface area as one 6 or 24 well, or three 60 mm plates.
Concentration is not all that matters. Another reason that a straight dilution is problematic is because it does not take into account the surface area to be coated. Your calculation needs to be based on mg per surface area, not a volume to volume ratio.
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