Etchevers:Notebook/Genomics of hNCC/2009/03/30
|Genomics of human neural crest cells||Main project page|
Previous entry Next entry
Froze what was left of R1113 cephalic cells: 113K in 1 mL Rich 90%/ DMSO 10%. Brought down in isopropanol to -30 then -80 and finally into liquid nitrogen at 5:30.
Fed the R1066 cephalic, passage 23 and R1066 trunk, passage 26 cultures, still sparse in 35 mm dishes. However, heterogeneous groups of cells, some small and numerous, others large and spread or even with those odd spaces in the cytoplasm as if they were flowing around something on the plastic. Ask Steph or Dawiyat to feed them on Thursday.
Manually removed the now perfectly round spheres on coverslips 1 and 3 of the attempts at neural differentiation. Subjected two of these to 5 minutes trypsin (rinsed in PBS, maybe lost one) and manual dissociation in 10% serum/DMEM before spinning down and resuspending in 6 mL Stemline N + B27 + EGF + retinyl ester - still the same lot, so vitamin A is probably long depleted. Spread out over some 34 wells, approximately 180 μL per well. At least 3 and at most 8 cells per well (This is well below commonly used measures of "clonal density"). See how they (may) grow over a week.
Fixed the three coverslips 2h in 4% paraformaldehyde. Rinsed extensively in PBS, left in PBS and filled adjacent wells also. Parafilm and placed in top right shelf ON DOOR of right hand refrigerator. Do beta-tubulin when you get back and perhaps Ki-67.
Cf. Singec et al. 2006 for both the neurospheres and immunohistochemistry. It would be nice to find some GFAP as well.
Tried to back up hard drive of other computer which crashed. Matthias T. helped me out immensely with this, so I have at least my personal folders.