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

  • Denhardt’s solution 50x D2532 5 mL 87.00 52.34
  • Cocktail inhibitor Phosphatase 1 10ul/1ml (cautharidin, bromotetramisde, mycrocystin LR in DMSO solution)

Sigma, cod. P2850 P2850-1ML 50.60 recommended as 100x 52.36

  • Cocktail Inhibitor Phosphatase 2 10ul/1ml (Na orthovadanate, Na m lybdate, Na tartrade, imidazide in H2O)

Sigma, cod. P5726 P5726-1ML 50.60 recommended as 100x 52.36

  • Pepstatin A 10ul/1ml P4265-1MG 31.20 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.

  • Cocktail inhib. Protease 5ul/1ml (AEBSF, EDTA, aprotinin, bestatin, E-64, leupeptin in DMSO solution)

Sigma, cod. P8340 P8340-1ML 38.70 52.36

  • 10% sodium deoxycholate D6750-10G 13.80 52.36
  • 0.1 mg/ml cycloheximide C1988-1G 99.10 or C4859-1ML 84.40 (100 mg/mL in DMSO) 52.34
  • Igepal CA-630 I3021-50ML 30.20 52.34
  • Triethanolamine 90278-100ML x2 bouteilles 37.10 52.34
  • Stemline Neural Stem Cell Expansion Medium (urgent!!) S3194-500ML 124.50 52.41
  • Laemmli Sample buffer 2x S3401-10VL 6mL 26.80 52.34
  • Tween 80 P4780-100ML 14.90 52.41
  • Retinyl acetate R7882-1G 29.90 52.41

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)

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