Etchevers:Notebook/Genomics of hNCC/2008/09/03

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 * style="background-color: #EEE"|[[Image:C14.jpg|128px]] Genomics of human neural crest cells
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 * style="background-color: #F2F2F2" align="center"|  |Main project page


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Sending myself hNCC
Candice sent yesterday the following vials:

- 1 ampoule de CCN-trunk R1066 passage 20 congelée le 27/12/07 - no cell # noted - 1 ampoule de CCN-cephalic R1066 passage 17 du 08/08/08 - 1.8 x 10^6 cells, this counting before freezing did not thaw well back in May

I have made up a new lot of 100ml medium (see protocols) with the old tested serum aliquot, as soon as cells are clean will divide them into different media to see how they get on. Used the Hovnanian current lots of T3, EGF and hydrocortisone.
 * Alethea 06:53, 3 September 2008 (EDT):

Procedure for thawing cells

 * Preheat 10 mL of medium in 37 °C waterbath in a 15 mL conical tube per aliquot of cells.


 * Thaw cells by holding cap in fingers and gently shaking within the waterbatch until most thawed but there is still an ice cube in the middle (a minute or two)


 * Bring both recipient and cells over to GSM hood, having wiped down with sterilising agent (EtOH 70% or better, quaternary ammonium-containing spray)


 * Uncap cells and dump over uncapped conical tube.


 * Draw up some warm medium and rince down the original vial, and add it to tube.


 * Spin tube in balanced swing-bucket rotor for 5-10 minutes at 1200 rpm.


 * Aspirate DMSO-containing supernatant and resuspend cells in warm culture medium (1.5 ml).


 * Place in Collagen-I-coated 35 mm dishes in a clean 10 cm non-coated dish, at 37 °C in incubator (5% CO2).

I also found that 2 10cm plate cultures of R1064 (passage 11 from 10-6-08) still had a few (apparently) live stubborn cells in them, even though the last medium change goes back to early July! Morphology much like what I had photographed before, perhaps I will put the photos up somehow. Fairly vacuolar, but don't dead cells all explode? (there was a fair bit of debris). So changed 2/3 of the medium for the fresh medium I just made up, see if I can't kick-start them into dividing again. But quite different cell shape from initial hNCC. Far bigger, to start, lots of actin filaments.


 * Alethea 07:27, 3 September 2008 (EDT):


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