Etchevers:Notebook/Genomics of hNCC/2008/09/04

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Passage of hNCC cephalic and trunk-level cells from embryo R1066

Both cultures had survived with very little cell debris in the 35 mm dishes. A little more death in the hNCC-T (trunk level), though less cell-dense than the C (cephalic level).

  • Rinsed in 5 ml warm PBS
  • Warmed trypsin aliquot, applied 0.5 ml for 3 minutes. Watched under the microscope, large plaques of dense cells detached.
  • Rinsed dishes to detach last cells with 3.5 ml warm PBS (no Ca/Mg). Transferred into 7 ml warm Rich medium (the one in the protocol).
  • Spun at 333 rcf (I think this is "g") = 1200 rpm in this Heraeus rotor, RT for 5 minutes. This was probably a bit too strong, the pellet was difficult to resuspend.
  • Counted each culture by diluting 1/2 in the 1x trypan blue that's available over the microscope.

- T: 35, 37 and 35 cells in square part of micrometer; this is in theory x 104 cells/mL. 2 or 3 dead cells per field tops.

- C: 102 and 105 cells in same part. x 104 cells/mL

  • Multiply these figures by 2 for the dilution and 0.5 ml at the time of dilution, to get a total of 3.5 x 105 cells for T and 1 x 106 cells for C cultures. This is a little odd because I thought there were to start a million cells in T and it was recorded 1.8 million cells in C originally on the tube.
  • Had added 2 more ml of Rich medium to each tube for a total of 2.5 ml each.
  • Decided to seed 2 x 105 cells per 35 mm dish to be not too dense or too sparse over weekend. Wanted to start testing the following samples of media:

- Panserin C6000 (cat P04-71600 for 100 mL) which is actually protein-free CHO medium, could be a - control (it's not what I requested, I think I wanted to give the 415 a shot for osteoblast differentiation)

- Stempan (cat P08-50500 for 500 mL but they gave me 100 mL and it's not obvious in the Dutscher catalogue)

- Panexin serum substitute (P04-95030) - thawed 10 mL aliquot lot no. 122205 exp 12/08, made 2 x 50 mL DMEM/F12 with Glutamax and 5% Panexin and 1x p/s for regular use, and one 50 mL aliquot of 10% Panexin in DMEM/F12 in case I want to have a higher % like 8% - PAN Biotech writes 10% is too much for a certain cell type. Once diluted, good for 1 month in theory.

- Panserin 401 cat P04-710401M for 100 mL "all cell types"

- continue in Rich medium

  • Divided the T cells in two for approximately 200,000 cells in two dishes, with 1.25 ml each and added in 1.25 ml Rich medium into one (R) and 1.25 Panexin-supplemented F12/DMEM in the other (P).
  • Divided the C cells in five for approximately 200,000 cells in five dishes, with 0.5 ml each, added in 0.5 ml R, P, Panserin C6000 (C), Stempan (S) and Panserin 401 (401) for 50% foreign medium O/N.

--- Lab meeting, see notes from conferences entry.


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