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Materials and Methods section:Confusion regarding volume of medium and number of EBs

Posted by sayandip on 01 May 2013 at 14:33 GMT

Dear Authors:

I am keen to follow your protocol for generating megakaryocytic progenitors from hiPSCs. My question is with regards to the following text:

"After 10 days, 72 EBs were transferred to each well of a 6 well flat-bottom tissue culture plate containing 7.2 ml of BPEL supplemented with 20 ng/ml rh thrombopoietin (TPO), 25 ng/ml rhSCF and 25 ng/ml rh interleukin (IL)-3 (Peprotech)."

72 EBs in 7.2 ml of medium sounds too much for ONE WELL of a six-well plate! Can you please confirm that this is true? In the Ng ES Nature Protocols paper which you have followed, they transfer 30 EBs in 3 ml of APEL medium per well of six-well plate on day 10.

I would be grateful if you can kindly reply to either sayandip@gmail.com or sayandip.mukherjee@ucl.ac.uk.

Thanks and Regards,
Sayandip Mukherjee.
UCL, London, UK.

No competing interests declared.