Etchevers:Notebook/Genomics of hNCC/2009/07/03
|Genomics of human neural crest cells||Main project page|
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Finding the cells in vivo
As we only ever see what amounts to debris after 7 days post injection, we decided to try again using the currently cultured DRG cells, which are wildly dividing again in the 150 cm2 flasks.
Have stained 30 minutes with CMFDA 1:1000 after diluting two vials into 10.76 μL DMSO, warming to 37°C, then placing into 2x 10.76 mL warm PBS. Rinsed flask for EMB090005 DRG cells p8, replaced medium.
Will aspirate, then replace with Rich medium for 30 minutes. Rinse with PBS, trypsin, spin down and count.
Meanwhile, make up more dilute collagen:
Diluted 78x (1.92 mL) into 150 mL 0.01M acetic acid (9 mL glacial) in MilliQ water, filtered into bottle. Then prepare a number of flasks for BWH group, 1h RT. (x10, of which 7 have been rinsed in PBS and remain in fridge).
Count for 0.5 mL cells = 1 square/25 = 58 cells. x25 x 104 = 1450 x 104 cells per mL. Made the mistake in calculations of multiplying by 2 instead of dividing, so was working on the basis of 29x 103 cells per μL, when in fact I had 7250 cells/μL.
Made up 129 μL to 150 μL with medium (added 20 μL) = "3.75 x 106 cells" but this is in fact 1/4: 937,500 cells in 150 μL. A 50 μL will inject 312,500 cells. (rather than the 1.25 x 106 desired).
The other dilution was supposed to be 150K cells in 150 μL, so I used 5.5 μL and made up the volume. However, in reality this is equivalent to only 37,500 cells in 50 μL. Which sort of stinks. And I threw away plenty of cells - is it possible to recover them since they are sealed? add in 17 more μL at least? Did this. So should have 160K cells now in 167 μL, and will inject 50K cells in 50 μL.
Also added back in 129 μL to the concentrated ones, but spun down 10 seconds at 2K g and removed same volume of supernatant, and resuspended by flicking. Now have 1870500 cells in 150 μL, which will lead to the injection of 623,500 cells in in 50 μL.
Rabbits pre-medicated 10:15, anaesthesia at 10:45 for injection before 11AM. Return for euthanasia at 12:15 to start fixation by 12:30. One rabbit per dilution, inject both corneas and maintain anaesthesia until end. One eye fix in buffered formalin, the other in 4% paraformaldehyde. (4 vials then).