I need to prepare conditioned medium for Jurkat cells.
I understand that it is medium in which the cells have already grown, and the cells filtered out, but how exactly should it be made?
- What concentration of cells should be seeded into the new medium
- How long should the cells grow in the medium/to what density before filtering
Cells are grown in the following:
RPMI 1640 supplemented with:
10% FBS
1% Glutamine
1% Sodium pyruvate
(Should I include non-essential amino acids?)
This medium is to be used for limiting cell dilution after electroporation with RNPs.
Hi Nikki Schütte . Usually, the way in which you are going to prepare the conditioned media depends also on the cell line you are using and the final purpose. I help you to have a general idea by providing you with two examples of conditioned media used for organoid culture:
-Rspo1 conditioned media. Usually, in this case, you seed your cells in T175 flasks with growing media and wait to have them confluent. At confluency, you carefully remove the media and start the conditioning. This means that you add fresh media (AdDMEM+hepes+glutamax) and keep the cells in the incubator for 1 week. After this period you take the media, centrifuge it and sterile filter it.
-Wnt conditioned media. You seed the cells in T175 flasks and start immediately with the conditioning. Therefore, you add the growth media and wait for one week. Then you take the media, centrifuge it and sterile filter it.
Hope I helped
Giulia
Hello Nikki Schütte
1. Culture Jurkat cells in an appropriate sized flask (say T-75 cm^2 or T-175 cm^2 flasks) under normal culture media (RPMI 1640 + 10% FBS + 1% Glutamine + 1% Sodium pyruvate) until optimal confluency. Please note that the cell confluency (80-100% confluency) will depend on the cell type such that it preserves the phenotype with minimal cytotoxicity or cell death.
2. Centrifuge the cell suspension at 1000rpm for 10 mins and aspirate off the normal growth medium (RPMI 1640 + 10% FBS + 1% Glutamine + 1% Sodium pyruvate), wash the cells 2 times with sterile PBS, then 2 times with serum-free RPMI 1640, and replenish the cells with serum-free RPMI 1640 media. You should preferably use serum- and phenol red-free media (20ml / T75 cm^2 or 30ml /T175cm^2 flasks).
3. After 24-48 hours (or other optimal time point) incubation in serum-free RPMI 1640 media, you may centrifuge the cell suspension and collect the supernatant (conditioned media), and sterile filter the conditioned media using 0.2-micron filter. You may use the conditioned media as is by dispensing 30 ml aliquots into sterile 50 ml tubes or you could further concentrate the conditioned media for example, by ultrafiltration or lyophilization depending on your application.
Please note: Non-essential amino acids is not required.
Best.
Hi Malcolm Nobre
I just want to make sure, how come do you transfer the cells to medium containing no FBS? What is the effect of this?
Hi Nikki Schütte
FBS consists of various proteins like growth factors, cytokines, and other soluble mediators. Since one would be interested in certain proteins in the conditioned medium produced by the specific cell-type that would affect the cell phenotype, adding FBS to the culture media during the process of collecting the conditioned medium would not serve the purpose because the proteins present in FBS would either produce an added effect or inhibit the effects of the conditioned medium.
Therefore you need to wash the cells with sterile PBS to get rid of FBS and use serum-free RPMI 1640 for either 24-48 hours or any other optimal time point when you need to make conditioned medium.
Best.
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