Etchevers:Notebook/Genomics of hNCC/2008/09/08

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Passage of hNCC-T and hNCC-C in different media

Observations:

  • R1066 hNCC-T are highly dense, crowded up against one another in whorls, in both the Rich and the Panexin-supplements (P) medium.
  • The 2 x 105 R1066 hNCC-C are now pretty tight, perhaps 95% confluent in areas, in the Rich medium and less so but still covering most surface in the P medium. However, you can tell the individual cells apart in the latter.
  • The 2 x 105 R1066 hNCC-C in Stempan (S) medium are maybe 80% confluent. They are fairly uniform, not too big but some cells have a few projections, and there is some cell debris not seen above.
  • The 2 x 105 R1066 hNCC-C in the Panserin 401 medium have more cell death, a fair bit of debris floating, but then again more cells, perhaps 90% confluent. The cells are still quite uniform, but rather rectangular, with more phase-dense endoplasmic reticulum around the centered nuclei.
  • Finally, the 2 x 105 R1066 hNCC-C seeded into the panserin C6000 medium (C) are suffering the most. They look more cobblestone-like, but not touching by much as they are not very spread; dark ER; most cell death but yet there are still lots of cells covering the bottom (75-80%)?



Decided to pass the trunk-level and cephalic-level cells in Rich and P media, and maybe the S/C media for the hNCC-C tomorrow.

3' warm trypsin after rinsed in warm PBS. Stopped in 6 ml 10% serum-containing RPMI, but maybe next time should just put into large volume warm PBS as they stuck a lot, particularly those from the P medium.

Cell counts:

hNCC-C in Rich: mostly small diameters, 9 to a square with a few 4 to a square or even a couple of big blasts 2 to a square; 29, 21, and 23 cells. x104 cells, is equivalent to 2.4 x 105 cells.

hNCC-T in Rich: 25, 24 and 30 x104 cells, is equivalent to 2.6 x 105 cells.

hNCC-C in P: Clumpy in tube, but did not see these clumps in Neubauer. 13, 13, 13, 14 x104 cells, is equivalent to 1.3 x 105 cells.

hNCC-T in P: As above. 19, 18, 12, 14 x104 cells, is equivalent to 1.6 x 105 cells.

Well, shoot me. Where did all those cells go? Divided the Rich medium into 2 x 35 mm plates, and put all of the P medium cells back into 1 x 35 mm.



In other news, here are the raw tag counts (divide by 50,500 and multiply by 106 to get LongSAGE tag counts per million for the hNCC column at least) of all genes I could find associated with chemokines or their receptors. I don't remember off the top of my head how many tags were for the other four SAGE banks - this is not yet published and in collaboration - but upwards of 35K, I think. These banks are established from microdissected bits of the neural tube from which the hNCC migrate at stages Carnegie 12 and 13, in brain and anterior spinal cord fragments.

Image:Chemokines NCC NT.xls

  • Alethea 09:35, 8 September 2008 (EDT):
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