Etchevers:Notebook/Genomics of hNCC/2008/10/08
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Plating cells on glass coverslips
I removed all cells from their surfaces, counted them (will have to add in counts that are currently written on a scrap of paper**, but were in the many tens-of-thousands or perhaps 100000 per well) and re-seeded them in 4 (or 5, for the S cultures) wells with a collagen-coated, UV-sterilized coverslip at the bottom.
The collagen had been a little polymerized, but it clearly did so more once dissolved into the 30% EtOH I prepared in the hood as per the day before. Put on 200 ul overnight, it was dry in the AM and they came unstuck with some PBS. Transferred with tweezers into a new plate.
Put 500μL of medium on each well: hEScGro cephalic, trunk cells (one in lower right corner); C6000; Stempan; Panexin 401. Took some of the 401 cells (1/3) and replated in 2 x 35 mm plates: one with pure 401 medium, and one with 4/5 Rich medium.
The 401 cells were R1066 CCN-C now p20 from 9/9, had been subjected to the attempt with the serum-containing fluorescent dye. So the DMSO doesn't hurt.
The H cells were R1066 ceph now p24; trunk now p27 from 22/9.
The C cells were ceph now p19 from 4/9, originally 2x105 cells.
The S cells were ceph now p20 from 9/9.
H-T (4 wells) 70/5 x 2 = 280 000 cells or 70 000 cells per well
401 (4 wells) 84/2 x 2 = 840 000 cells
C6000 (4 wells) /5 x 2 = 192 000 cells or 48 000 cells per well
S (5 wells) 86/3 x 2 = 573 000 cells or 114 600 cells per well
H-C (4 wells) 58/5 x 2 = 232 000 cells or 58 000 cells per well
....401 medium 140 000 cells in 6 parts? and seeded two into 35 mm dishes, the other 4 onto coverslips