Effectively washing adherent cells? - how do I optimize washing? (Aug/21/2007 )
Hi all,
I am culturing adherent neuronal cells in a T25 flask - The problem is that I need to remove
all "contaminant" proteins that are in the cell culture medium, in order to do the follow up-experiments.
I usually wash 3 times with 1-2 mL buffer (containing CaCl2, NaCl, and HEPES, pH7.4) and swirl a bit in between,
but in my results I still have major contamination of medium proteins. It is important that the cells remain adherent
in the flask for further treatment.
Do you have any tips on how I could optimize the washing? Longer incubation while washing, or maybe different buffer
composition to effectively lure these proteins out of their hiding place? 
hope you can give me some advice!
More buffer!
How much medium do you have in the flask normally? I bet more than 1ml. So to wash it off, you have to add at least the same amount if not more. I would use about 8 ml per wash, just gently rock the flask for a few seconds, pour out and wash again.
Depending on how adherent your cells really are, you might loose some if you're not careful.
LeserattePD
I am culturing adherent neuronal cells in a T25 flask - The problem is that I need to remove
all "contaminant" proteins that are in the cell culture medium, in order to do the follow up-experiments.
I usually wash 3 times with 1-2 mL buffer (containing CaCl2, NaCl, and HEPES, pH7.4) and swirl a bit in between,
but in my results I still have major contamination of medium proteins. It is important that the cells remain adherent
in the flask for further treatment.
Do you have any tips on how I could optimize the washing? Longer incubation while washing, or maybe different buffer
composition to effectively lure these proteins out of their hiding place?
hope you can give me some advice!
Absolutely! Use at least the same amount of medium normally found in the flask or 2-3 mLs more to wash effectively!
How much medium do you have in the flask normally? I bet more than 1ml. So to wash it off, you have to add at least the same amount if not more. I would use about 8 ml per wash, just gently rock the flask for a few seconds, pour out and wash again.
Depending on how adherent your cells really are, you might loose some if you're not careful.
LeserattePD
I am culturing adherent neuronal cells in a T25 flask - The problem is that I need to remove
all "contaminant" proteins that are in the cell culture medium, in order to do the follow up-experiments.
I usually wash 3 times with 1-2 mL buffer (containing CaCl2, NaCl, and HEPES, pH7.4) and swirl a bit in between,
but in my results I still have major contamination of medium proteins. It is important that the cells remain adherent
in the flask for further treatment.
Do you have any tips on how I could optimize the washing? Longer incubation while washing, or maybe different buffer
composition to effectively lure these proteins out of their hiding place?
hope you can give me some advice!
Thanks for your advice ! Seems kinda obvious all of a sudden 
How much medium do you have in the flask normally? I bet more than 1ml. So to wash it off, you have to add at least the same amount if not more. I would use about 8 ml per wash, just gently rock the flask for a few seconds, pour out and wash again.
Depending on how adherent your cells really are, you might loose some if you're not careful.
LeserattePD
I am culturing adherent neuronal cells in a T25 flask - The problem is that I need to remove
all "contaminant" proteins that are in the cell culture medium, in order to do the follow up-experiments.
I usually wash 3 times with 1-2 mL buffer (containing CaCl2, NaCl, and HEPES, pH7.4) and swirl a bit in between,
but in my results I still have major contamination of medium proteins. It is important that the cells remain adherent
in the flask for further treatment.
Do you have any tips on how I could optimize the washing? Longer incubation while washing, or maybe different buffer
composition to effectively lure these proteins out of their hiding place?
hope you can give me some advice!