Etchevers:Notebook/Genomics of hNCC/2009/03/27
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Animal injections and neural differentiation
Fed cells yesterday, cleaned up debris. Lots of cells in R1113, but they look dominated by large, fiber-containing cells with serrated edges. The rabbit corneal endothelial cells, in passing, are not proliferating - they have spread, but are too far apart on the plastic surface.
Note that some others have experienced CMFDA toxicity although that was on exposure to light and creating free radicals (vitamin E seemed to protect against it).
CMFDA manual here.
Review of previous trial:
Some use 10 μM final concentration; the 25 μM last time was too much and I was thinking of using 5 μM (2000x dilution). But 10 is easier (1/1000).
Chose 10 μM final concentration for one T25 flask.
This was incubated 35 minutes in DMEM as diluent, then let cells recover 30 minutes in Rich medium. Trypsinized and resuspended in 2 mL tube with 0.412 mL medium for 100 μL putative injections.
Cell count was for 232K cells total, approximately 50K cells per 100 μL injection.
The three rabbits, Stephane noted which received what, but Emilie carried out the injections using the new 250 μL Hamilton syringe that I had sterilized in 100% EtOH for 45 minutes, rinsed in PBS, and then backfilled in PBS. Need to curve the 30.5 gauge needles such that the bevel is up, not up and curved to the left as I thought would be most convenient.
Pre-anaesthesia at 9:30. Real anaesthesia at 11AM. No movement or reaction at all of any of them. Proceeded with glove to isolate eyeball and antibiotic vaseline as the previous trial.
Emilie might be available also for neural tube microdissections and culture in the future.
A really clear phenotype - the balling up of the cells I had seen in the low FGF2, high EGF, retinyl ester-containing B27+Stemline neural medium is confirmed.
Even more interesting, of the 6 remaining coverslips that had been in that medium, I changed the medium of 3 of them, two of which had the balls, back to the B27+Stemline neural medium with high EGF but the FGF2 concentration used in Rich medium, and no retinyl ester.
Nearly every cell in these is stone dead, like the following row had been when I had seeded the wells to begin with earlier in the week.
Find out about neurosphere propagation now, perhaps the cell aggregates are neurospheres (some are round, others are like rolled-up edges). All three remaining coverslips have them. Fix these on Monday, but perhaps remove "spheres" for further propagation, first.