I'm planning to make conditioned media from various cell lines for some experiments and have come across various protocols, which all differ in their methods.
My current plan is:
1) Culture cells in T75 flask until 80% confluency.
2) Once at 80%, I'll aspirate the media, then wash 2x with serum-free media.
3) After washes, add 20 ml serum-free media and leave flask for 48h
4) Harvest the media, centrifuge and freeze down as 1 ml aliquots (I want to use these CM aliquots in migration/invasion assays).
My main question is 20 ml to large a volume or should I concentrate this down to a smaller volume?
Thanks in advance!
Hello David J Walker
The purpose of the conditioned media is to use the beneficial effect of the conditioned medium of one cell type on another cell type.
20ml volume of conditioned media may be aliquoted into smaller volumes (say 1-2ml each) and frozen at -80 degree C for later use. You need to note that even when strict precautions are followed and the protocol is optimized, handling the cells in vitro results in cell necrosis, and the intracellular proteins released by necrotic and apoptotic cells may also add up along with the desired secreted proteins.
On the other hand, secreted protein concentration in the conditioned medium is usually low and the desired secreted proteins are in a soluble form. It would be desirable to retain solubility during the concentration step, and use those methods that do not involve precipitation, for instance centrifuge filtration, dialysis, or evaporation. Centrifuge filtration with micro concentrators can result in protein binding to the membranes (even when low-protein-binding membrane devices are used), and the proteins of interest could be lost. During dialysis, the protein can precipitate because of low salt concentration in the dialysis buffer. You could use Speed Vac concentrator to concentrate conditioned medium.
But all of these above methods are going to alter the proportion of various molecules, and may not specifically increase the concentration of the secreted protein of interest which is going to serve the purpose.
So, I recommend that you harvest the media, centrifuge to remove the non-adherent cells and prepare aliquots of 1-2ml of the conditioned media, as you mentioned, and store these aliquots in -80 degree C for future use in migration/invasion assays.
Best.
20 ml of serum-free media is a reasonable volume to add to a T75 flask containing cells that are at 80% confluency. This should allow for enough media to be produced by the cells, while also preventing overgrowth of the cells. However, it is important to note that the final volume of conditioned media will depend on the specific cell line you are using.
You may want to consider concentrating the conditioned media to a smaller volume before freezing it, as this will help to save storage space. Additionally, concentrating the media will also increase the concentration of any secreted factors, which may be beneficial for your migration/invasion assays.
Common methods to concentrate the CM are centrifugation and filtration.
- Centrifugation can be done using a centrifugal filter unit with a molecular weight cutoff of 10-30 kDa, which will allow you to remove any large debris or cells while retaining the smaller molecules of interest.
- Filtration can be done using a sterile filter with a pore size of 0.22 micron which will remove any bacteria or other contaminants.
Both methods will allow you to achieve a higher concentration of the secreted factors of interest in the conditioned media.
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