Etchevers:Notebook/Genomics of hNCC/2009/04/02

Genomics of human neural crest cells

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Sacrococcygian teratoma from St Etienne Stillborn infant that had died on Monday or Tuesday from the vascular problem, surgeons removed seven samples of 8 cm3 from large SCT and sent in RPMI. These samples were numbered 1-7. Alexis has the paperwork. 1 = coccyx, 2 = close tissue to coccyx. 3-7 various cystic and fibrous areas. The cysts are very tough; I tried to take some of blocs 1-3 to dissociate. First cut the coccyx in 1/2 along midline, fixed 1/2 in 4% PAF and removed some tissue in the cartilage to dissociate. The nearest face of block 2 to the coccyx was signaled with a piece of suture. Again cut in 1/2 so the proximal-distal axis was respected in both 1/2s, and fixed 1/2. Block 3, large cysts in both 1/2. Then further dissociated the tissue - did not cut small enough in the end, as only about 200K cells were freed from large tissue chunks. Put to incubate about 1h in either PBS + 250ug/mL Dispase + 1/100 collagenase = DC, provided by Andrea in MCC's lab; or trypsin-EDTA, in 6-well plate. Candice did all further trituration and processing. The rest went back to Alexis to be frozen in cryostat block or cryotube for later RNA extraction. Because so few cells were isolated after enzymatic digestion, I told her to seed all into a 6-well plate rather than freeze down the enormous quantities needed to do a cell sort next Monday. Tomorrow, trypsin-treat and pass into 25 cm^2 flasks, if relevant after rinsing away dead cells. If they still do well by Monday, save perhaps a few to continue to propagate/freeze? and the rest can go into a cell sort programmed at 1PM using SSEA-4 under the cosupervision of Andrea and Isabelle. Then Alexis got his mouse protocol approved by N Stadler and can inject next week into one or both of the nude mice provided kindly by the 7th floor of Lavoisier in subcutaneous, in company of Sabine. Placed Invitrogen order for B27 and other products, including Sigma Retinyl acetate again, and Tween-80 to help in dispersion. Need to write up protocol for Candice for the dilutions. Apparently the stocks can not encounter air/O2 at all - being an antioxydant of course. The idea is to force neuronal differentiation so that maybe Phox2b will work. Using two cell lines, 8 wells - top row = regular medium, bottom row will use neuronal medium if available. But since she wants to take the week of Easter off, this won't fly. That was inconvenient. But the Sigma order hasn't even been placed yet. Ordered more LabTeks, too.