Etchevers:Notebook/Genomics of hNCC/2009/04/06
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Rabbit EC, hNCC, Sigma order for Necker
1.5h endothelium + Descemet membrane from last Friday's dissection in 2.4U/mL (PBS) Dispase II cf cat 11276921-001 de Roche, thawed from freezer, old stock. 37ºC. Triturated with flame-polished Pasteur pipette. Rinsed in 20% serum/DMEM high glucose-Glutamax, then cultured in this medium in same wells.
Passed R1066 cephalic and trunk cells; only counted 40K and 80K cells total, which seems very low. Instead of putting them on collagen-coated flasks (PBS, from last week in fridge), decided to reseed in 35 mm dishes in fresh Rich medium.
Also took 10% of the cephalic cells (4K in theory) and seeded into one of these flasks, in fresh neural differentiation media. Shook up retinyl ester-in-DMSO, pipetted 10ul into 10 mL PBS, then added 50 ul of this to 50 mL of Stemline neural + 1x B27 (kept in fridge since last use).
Ordered Sigma reagents: Biologie moléculaire = 52.34, biochimie = 52.36, culture = 52.41
Sigma, cod. P2850 P2850-1ML 50.60 recommended as 100x 52.36
Sigma, cod. P5726 P5726-1ML 50.60 recommended as 100x 52.36
needs to be dissolved at 1 mg/ml in 10% (v/v) acetic acid in methanol (9:1 methanol:acetic acid). Solutions of Pepstatin A can be heated as high as 60 °C without any decomposition of the peptide. Stock solutions at 1 mg/ml are stable at least a week at 4 °C. A 1 mM solution in methanol or DMSO is stable for months at -20 °C. If solutions become darker yellow, the reagent is hydrolyzing.
A working concentration of 1 μM is stable for at least one day at room temperature.
Sigma, cod. P8340 P8340-1ML 38.70 52.36
1g of retinyl acetate, ref R7882 (lot 047K07091) in 3.28 mL of DMSO at 60°C = 1M (328.49 g/mol). Want 1 μM and B27 has 0.61 μM already. Aquasol A has 12% Tween 80, for 15 mg/mL (286.45 g/mol). = 50mM = 50,000 factor dilution to get 1 μM.
--Heather 09:53, 7 April 2009 (EDT)