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.
- 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.