Etchevers:Notebook/Genomics of hNCC/2008/10/07
|Genomics of human neural crest cells||Main project page|
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Feeding cells, preparing coverslips
Fed all the cells as Steph did last Friday, presumably, with their own media (S, C6000, 401, H on trunk and cephalic). The C6000 are less cobblestone-like than they had been and more like the others, though many cells had detached.
Found some glass 14mm coverslips to place in 24 well plate. Sterilized one side then the other under UV for 30 minutes (first in the plastic of plate, second on the top of the plate, using sterile forceps to manipulate.
Will place in wells and coat with collagen, then transfer to new plate tomorrow. Collagen Millipore/Upstate catalog #08-115, lot# DAM1471400, is formulated with 100 mg in 18.9 0.02N acetic acid, which is 5.3 mg/mL.
Want to use at 2.5 μg/cm2 according to recipe using EtOH 30%. The coverslips are 1.54 cm2 so that means need 3.85μg/coverslip. If foresee 200 μL x 25 this will be in a volume of 5 mL, of which 1.5 mL should be of 100% EtOH. And want 96.25 μg of collagen in there, at a concentration of 5.3 μg/μL, which means you have to use 18.2 μL of collagen solution in those 5 mL.
Then cover surface with 200 μL of preparation and allow EtOH to evaporate under hood overnight. Cover and store at 4°C. The other option would be to dilute to 5-10 μg/cm2 in PBS, cover surface, incubate 10-30 minutes, aspirate and rinse with PBS/medium. I planned to rinse the EtOH coverslips before use as well. Although the medium has HEPES buffer, probably not necessary.
Worked on Puting's C12 figure, now just looking for size bars.