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gnommiAs I spent most of last week trying various strategies to assess the adhesion-free growth capabilities of my cell lines with middling success, it is time for Plan B. Back to dynamic viscosity pondering, Batman!
Ruled out are
Serum-free media (removes attachment factors and matrix proteins present in serum-containing medium): works for one line, but the other two lines stick down anyway.
Serum-free media + polyHEMA coated plates (polymer dissolved in acetone:alcohol then used to coat dishes, forming a hydrophobic non-adherent layer): works after a fashion but unpolymerised polyHEMA dissolves in medium after a few days to leave uncoated regions of dish to which the bloody cells stick.
Ergo: Time for methocel. Freshney (ALL HAIL THE GREAT FRESHNEY: and see p226) suggests layering sterile agarose or Noble agar (how do I get this, practically?) into dishes and allowing to set, then diluting cell suspension in 1.6% 4 Pa-s/4000 cps methocel diluted 1:1 with 2X growth medium on ice, adding it to the plates and allowing to set (as much as methocel does) at 37C. You can see why I might have been avoiding this option.
The advantages of methocel cloning are that I can rule out the possibility of cells aggregating on plating rather than forming spheroids by proliferating and actually divine whether density drives spheroid formation via secreted factors or not.
Anyway enough of that. I am trying not to procrastinate again, but as usual I have no interest in whether there are stem cells in my lines or not, and the things I am interested in doing are of no interest to anyone else. Also, I had lime and coriander microwave rice with sweetcorn and crunchy peanut butter for breakfast. I think I need more sleep.
Ruled out are
Serum-free media (removes attachment factors and matrix proteins present in serum-containing medium): works for one line, but the other two lines stick down anyway.
Serum-free media + polyHEMA coated plates (polymer dissolved in acetone:alcohol then used to coat dishes, forming a hydrophobic non-adherent layer): works after a fashion but unpolymerised polyHEMA dissolves in medium after a few days to leave uncoated regions of dish to which the bloody cells stick.
Ergo: Time for methocel. Freshney (ALL HAIL THE GREAT FRESHNEY: and see p226) suggests layering sterile agarose or Noble agar (how do I get this, practically?) into dishes and allowing to set, then diluting cell suspension in 1.6% 4 Pa-s/4000 cps methocel diluted 1:1 with 2X growth medium on ice, adding it to the plates and allowing to set (as much as methocel does) at 37C. You can see why I might have been avoiding this option.
The advantages of methocel cloning are that I can rule out the possibility of cells aggregating on plating rather than forming spheroids by proliferating and actually divine whether density drives spheroid formation via secreted factors or not.
Anyway enough of that. I am trying not to procrastinate again, but as usual I have no interest in whether there are stem cells in my lines or not, and the things I am interested in doing are of no interest to anyone else. Also, I had lime and coriander microwave rice with sweetcorn and crunchy peanut butter for breakfast. I think I need more sleep.
