Etchevers:Notebook/Genomics of hNCC/2008/09/17

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Cultures - feeding and passing

C6000 cultures of cephalic cells: still cobble-stone like in 35 mm dish, still not confluent. Removed supernatant, spin down with the previous supernatant/contents of the 25 ml flask.

Also trypsinated the two R1064 dishes remaining few cells, used 15% serum to stop, this is now P12 but last one was on June 10th. Place on new 35 mm dish.

Looked at C and T cultures - the large dishes (including handmade collagen) are pretty confluent but the 35 mm ones are irregular. Put all dishes onto a 150 cm2 flask each (trunk, cephalic) for WE. Feed on Friday. Want to get lots of cells, split on Monday for some freeze, perhaps some small 35 mm dishes for trials with the CellTracker reagent. Inject cornea-opaque rabbits when? probably on Wednesday/Thursday next week.

  • Heather 03:53, 17 September 2008 (EDT):


  • C dishes: counted 62, 71, 56, 66 x 10 4 or 6.4 x 10 5 cells w/o trypan blue. Resuspended quickly in 1 mL, took sample from still moving. Cells are bigger, lots of 25-40 μm diameter. This means there was (a) a little cell loss from passaging and (b) no cell division in two days? Lots of doublets, though.
  • T dishes: counted 56, 44, 61 x 10 4 cells/ml or 5.4 x 10 5 cells total. Had treated as above. Sophie has also noticed low cell numbers for apparent confluency. Thinks that cell counting is "off". Do they really spread out much more on the surface? not so apparent when the cell membranes are touching, need to check nuclear density perhaps.
  • Plated in 20 mL each Rich medium. Not enough for pure Rich medium for next time, will have to gamble on the serum I just bought and cross my fingers.
  • Steph says that the rabbits have corneal opacity grade 2 and are ready to graft. Need to check cell labelling though. Or just choose lowest "high" concentration of CellTracker at 5 μM in F12/DMEM.

- Apply 30 minutes of staining solution in F12/DMEM, over adherent cells.

- Rinse and apply 30 minutes of R medium to let cells recover and modify the chloromethyl group on the dye so it stays in the cells

- Trypsinize and pellet.

- Resuspend in 200 μL per cornea want to inject. Transfer into sterilized low-adherence microcentrifuge tubes to bring to animal care facility.

  • Heather 04:58, 17 September 2008 (EDT):