Now I'm preparing L929 supernatant for BMDM cell culture later.
After harvesting the supernatant, everything seemed fine, I filtered through 0.22 filter then put it in the -20 oC fridge, at that time the medium still looked red, not turbid.
However, after frozen, it all turned yellow, then I wonder if it is because of the supernatant are too acid due to too many cells and lack of nutrient maybe. After full confluent cells in 75cm² flask, I change medium and 3 days after I collect the supernatant.
But the fact is only after frozen the yellow could be seen.
In the past, I used this kind of yellow L929 supernatant for growing BMDMs, and I had some problems with BMDMs, it grew in the form of activated ones somehow.
So now to check the L929 supernatant quality, I notice the yellow problem, do you think it will possibly affect the BMDM differentiation or activate it ?
Is there anyone having same experiences with this? Please share with me.
Thank you so much.
Hi Anh Thi Trang Tran . Are you storing the media in a frost-free -20oC? If so, it is constantly freezing and thawing and giving anything in the media a chance to grow. It can also alter the bicarbonate balance in the media buffer. For reusing media, it is best to store it at -80oC.
Did you thaw them yet? If not, thaw a vial and check if the reddish color is coming back.
Every time we froze cells in plating medium (containing the pH indicator) or medium by itself, it turned yellow. Upon thawing it reverted back to its original color. To determine if temperature would change the characteristics of the medium we were using, we tested medium only side-by-side taken from the same initial batch batch of medium. One set was frozen at -20C, one set was frozen at -80C, one set was frozen in liquid nitrogen (-196C), and the other set refrigerated at 4C. ALL media that was frozen (-20C, -80C, & -196C) turned yellow. When thawed, ALL frozen medium matched the color of the refrigerated medium.
We then tested each set of medium with different cell clones in our lab (the clones were generated by repetitive single cell clonogenic analysis using conditioned medium), e.g., telomerase positive totipotent stem cells, pluripotent stem cells, mesodermal stem cells; telomerase negative mesenchymal progenitor stem cells (MSCs); and representative differentiated cells (skeletal muscle cells). In every combination tested, we found no significant difference in plating density, propagation, recovery from collagenase release, viability, etc.
Results: Freezing (-20C, -80C, or -196C) changes the color of the medium from red to yellow, while thawing reverts it back to its original (red) color. Changes in the color of the medium does not change the characteristics of the medium for supporting growth of cells in culture.
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