Etchevers:Notebook/Genomics of hNCC/2009/02/20

Genomics of human neural crest cells

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Making new media Have received from Dutscher, the order for ref 500101ES, SVF special cellules ES "100 mL" but these were actually 6 x 50 mL bottles delivered. Still PAN sera, "FBS special designed for ES cells" from South America, lot P281803ES. Prepared completely new media, before passing the cells that Dawiyat fed this week. These are quite dense in the 35 mm dishes (9.6 cm2, and swirls of paths have formed. I have coated with homemade collagen-PBS four x 25 cm2 T25 flasks for 2h at room temperature, 3 mL each as per the Eccles protocol. I am trying to get the 1 g of retinyl acetate, ref R7882 (lot 047K07091) from Sigma, to dissolve in 3.044 mL of DMSO at 60°C, since it won't at room temperature. This should give me a 1M solution, from which I can dilute to 1 μM. Apparently a physiological concentration used in B-27 supplement, that is now removed in B-27 without retinyl acetate for NSC propagation, would be 0.61 μM (cf. Contin et al., 2006, who were kind enough to finally specify this as per the original article to which I don't have access). Meanwhile, I made up three other media: Rich medium. As usual, but using the new serum lot. Rich medium, but using the new serum lot, Stemline-Neural medium to replace the DMEM/F12, and supplementing with 1x glutamine (2 mM I believe), but no HEPES. The Stemline-Neural is the old one from last October since the new bottle has no specification for when it will finally arrive. I called this StemN-serum. Stemline-Neural + 1x N1 supplement, N6530 5 mL, lot 088K8409 from Sigma, 1x glutamine, 1x pen-strep, FGF2 and EGF in the same concentrations as the Rich medium. 50 mL of this. If I can get the retinyl acetate to go back into solution, I will take 25 mL of the above and add in a 1:1,000,000 dilution of the retinyl acetate-in-DMSO. I suppose I would first have to dilute 10 μL into 10 mL PBS, then take 25 μL of that dilution. And I would have to do the same but with DMSO, and add it into the Stemline-Neural/N1 above. The idea is to trypsinize the cells and re-seed 1/4 into each flask, with one of each of the above media. If the retinyl acetate does not go into solution, I will use the first three media and compare to the older lot of Rich diluted 1/2 with Stemline Neural, as all last week they were in that medium and quite happy. Also, the low-density cells look alright still, as well. Heather 12:05, 20 February 2009 (EST): Update: the DMSO-dilution of retinyl acetate works, though it dissolves the plastic in the membrane over the lid, as well. As it cools to room temperature, it crystallizes out again. First I tried pipetting 10 μL into 10 mL PBS, but it congealed half in the pipette and the rest on the walls of the Falcon 15 mL tube. Vigourous shaking did not get it all in solution, but it was cloudy. Then I tried again, but after having reheated the stock solution. Same effect (in 5 mL). Finally, I warmed the PBS, warmed the DMSO, and tried a 1/10 dilution - 0.5μL into 5 mL PBS. This looked like it might have smeared on the wall, but rapidly went into (transparent) solution. I then diluted 250 μL of this with 24.5 mL of Stemline-Neural + 1x N1 and called it "N1 + vit A". 5 mL of each medium with 125 μL of the cells, resuspended in 500 μL of Stemline Neural + serum for counting. I was vigorous about unsticking them from the dishes as they did not come up easily even after long trypsin treatment (and put Rich medium back on the 35 mm dishes to see what happens). HUGE cell counts! 365 x 104 cells per mL x 0.5 mL = 1825000 cells. Could have frozen some! But instead divided among the 4 flasks for 456,250 cells per 25 cm2 or 18,250 cells per cm2 seeding density. Cf. this entry. And try to remember to put the pipe after the link and before the words when it's an internal link, but nothing at all when it is an external link. Heather 13:28, 20 February 2009 (EST):