- will pass the S, C and 401 cultures into 10 cm plates to see if the cells are ready, and also because I have no more plastic ware. Could try manual coating but should still generate a fair number of cells if they take. Change medium of the R and P cultures.
- yesterday, asked Stephane to order BD Biocoat plastic including some large 150 cm2 tissue culture flasks; these to grow up lots of cells for cornal implant idea we've developed. For live cell labeling, have ordered Molecular Probes CellTracker for initial implants (membrane dye, will dilute after 4 cell divisions or so) (but see this article, PDF for Vybrant DyeCycle which does the same but in the nucleus; I was ill at ease using a DNA intercalator), and have also ordered an anti-Ki-67 epitope, which appears on cells undergoing proliferation (absent from G0 stage) and is reportedly human-specific, though I'd have to look up those references again.
- Alethea 02:10, 9 September 2008 (EDT):
- Also changed C6000 medium, lots of floating cells (but it is for suspension cultures) and debris. The old summer cultures still have their recalcitrant, "cystic" cells which have phagocytosed a lot of small debris in the dishes. Will be tight on Rich medium so did not change it.
- Stephane suggests trying to trypsinize them anyhow and concentrate in very small wells (manual coat 12 well plate? or 4-well Nunc?) to see if you can boost them again.
Showed Stephane where media are etc. Transferred all into 50 ml conical tubes for easy finding in the fridge.
When passed S (more "polyhedral" morphology, some debris) and 401 (more like R/P cultures, lots of fibers and membrane prolongations, largish cells, still heterogeneous, clearly nearly confluent) into 10 cm dishes, had 63 cells over 5 Neubauer fields (x2 x0.5 ml) or 1.26 x 105 cells in the entire dish for the S cultures; 1.16 x 105 cells for the 401 cultures. A real test of their proliferative ability. Will change medium on Friday.
- Added 0.5 ml fresh medium to R and P cultures. Transfer into 10 cm dishes tomorrow.
- Alethea 04:28, 9 September 2008 (EDT):