Etchevers:Notebook/Genomics of hNCC/2008/09/22

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Test of CMFDA toxicity

CMFDA is 5-chloromethylfluoresein diacetate, it has a MW of 464.85 and there are 50μg per vial (20 or so in the package). Warmed to room temperature, and diluted in 10.76 μL DMSO.

The calculation is have 50 x 10-6 g x 1000 mmol / 464.85 g = 1.0756 x 10-4 mmol in the vial. Want 10 mM final so dilute by 1 x 106μL / 10 mmol which yields 10.76 μL DMSO for a 10 mM solution.

Then dilute that solution to yield 5 μM which is 2000-fold.

Diluted 0.5 μL in 1 mL rich medium for 5 μM, 0.5 μL in 5 mL for 1 μM and 9.76 μL in 3.9 mL for 25 μM. SHOULD HAVE USED SIMPLE F12/DMEM - maybe the serum was problematic (this is hindsight relative to low fluo in photos).

Applied the following to these cultures after simply removing original medium:

  • 1 μM for the S-medium culture 10 cm dish
  • 5 μM for the C6000 medium 35 mm dish
  • 25 μM for the 401 medium 10 cm dish

Replaced in incubator for 30 minutes.

Aspirated and replaced ALL with Rich medium.

Cell counts after having passed the flasks were low last week; in the cephalic cultures, there were only 84 x 104 cells and in the trunk cultures, 60 x 104 cells. Had diluted in 8.4 ml for the former and 6 ml for the latter for 100000 cells per ml. Seeded three 35 mm dishes each for eventual trials of neural serum free medium (Sigma, if arrives) and Millipore's hEScGro medium (and Rich as comparison). The rest went back into large flasks T150. (Reseeded therefore 540000 cephalic and 300000 trunk cells).

  • Heather 03:10, 26 September 2008 (EDT):