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:

  • Warm CMFDA to room temperature, dilute in 10.76 μL DMSO, which is 10 mM.
  • Dilute in Stemline medium *with no serum* to 10.76 mL. Actually, protocol specifies "Avoid amine- and thiol-containing buffers." so perhaps just stick with PBS - don't my media have amines? Rinse one flask with same medium, replace with CMFDA, incubate for 30 minutes.
  • Aspirate and replace with Rich medium. Let the cells sit for another 30 minutes.
  • Trypsinize and resuspend in 2 mL tube with 0.2 mL Rich medium for 50 μL injections.

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).

  • Heather 02:05, 27 March 2009 (EDT):

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.

  • The first rabbit was injected with that originally curved needle, but she pierced into the aqueous humor. Still injected into stroma but not directly in scar this time. Some cells in, some out. Approximately 80 μL "in" the stroma but plenty in the anterior chamber as well.
  • The second rabbit, 100 μL seemingly into stroma, but two injections. Used properly curved needle.
  • The third rabbit, one injection where the cleavage plane was between epithelium/Bowman's membrane and the underlying stroma. Made for a much reduced spread of the volume, perhaps 90 μL made it in. Cell injection is cloudy, visible with naked eye.

Emilie might be available also for neural tube microdissections and culture in the future.

Last time we did this trial.

  • Heather 10:10, 27 March 2009 (EDT):

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.

  • Heather 10:28, 27 March 2009 (EDT):
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