FACS Aria Sorter - Concentrating the sorterd polyplex sample (Mar/12/2007 )
Hi all,
I am working on FACS : trying to sort the polyplexes (PEI/DNA) particles depending on their size distribution.
When we sort in FACS Aria Cell sorter the sorted sample will be diluted with sheath fluid in large volumes. Say 100ul diluted to 700ml. After sorting the sorted sample contaning polyplexes, must be added to cells to see further studies in our project.
Right now the problem is, how to concentrate the polyplexes (contains DNA labeled with FITC) ? I tried vivaspin method with different MWCO 30,000 & 10,000. Centrifugation I can not do as polyplexes will agreegate.
Please any body can suggest the way to resolve this problem. It woulb be great to discuss with people working in related study of Polyplex or FACS.
Thank you in advance, if u have answer.
I am working on FACS : trying to sort the polyplexes (PEI/DNA) particles depending on their size distribution.
When we sort in FACS Aria Cell sorter the sorted sample will be diluted with sheath fluid in large volumes. Say 100ul diluted to 700ml. After sorting the sorted sample contaning polyplexes, must be added to cells to see further studies in our project.
Right now the problem is, how to concentrate the polyplexes (contains DNA labeled with FITC) ? I tried vivaspin method with different MWCO 30,000 & 10,000. Centrifugation I can not do as polyplexes will agreegate.
Please any body can suggest the way to resolve this problem. It woulb be great to discuss with people working in related study of Polyplex or FACS.
Thank you in advance, if u have answer.
1. I am not sure if the methodology is valid and practical. You are essentially separating nanoparticles suspended in droplets, right? The droplet size will be the dominant factor if I understand correctly the principle of FACS. How do you tell the difference of polyplex say 1 um in diameter, distributed in 3 um size droplets verse 0.5 um polyplexes resided in the droplets with the same size? Are you trying to sepaprate highly diluted samples by intensity?
And for the practicality, how long did it take for you to sepaparate 700 ml sample?
Could you use a simpler method, such as differential centrifugation or centrifuge on sucrose density gradient to separate these complexes of different sizes?
2. Interionic complexes are not very stable by nature and the presence of excess of polymers are needed to stablize the complexes. The concentration of the free PEI will decrease after dilution and sepapration. and certainly you will lose more after ultrafiltration.
To avoid aggregation, you can use dialysis bag method with absorbant on the outside. Polyacrylamide powder should absorb water from the bag efficiently.