I have a doubt about conditioned media while collecting it. I do tissue culture and knockdown experiments and collect conditioned media from them. While collecting the media from the plate do i need to pippete up and down to collect all the stuff attached to the plate as i can see small things floating and some attached to the bottom of the plate??? what i usually do is i just collect the media without disturbing the stuff attached to the bottom of the plate and i spin the medi at 4C for 5-10 and at 1000rpm and when i check for the total protein concentration (bradford assay) i get very less amount of concentration. and based on this total protein concentration i need to use this gene KD media on my cells to check the effect on another gene that im interested in.
could you please suggest me some tips regarding this problem.
Thnaks in advance
If you are only concerned about some cytokine/protein that is being excreted into your media then you should just remove the liquid without disturbing they adherent cells. Depending on what you are looking for, you may want to filter your cells to remove any of the larger particles (dead cells) that could alter your overall protein concentration. From here, you could spin down your cells and isolate your protein.
Can you be a little clearer in what you are looking for?