dear collegues ,
i'm needing your help with the protocol of differentiation of MCS human , i want put to the media 50 ml l-ascorbique ACID concentration 50 micromolar , how can i calculate the volume take from solution L-ASCORBIC ACID to put it in my media ??
thx a lot for your help
Simply use the formula od concentration: C=n/V and do a rule of three.
Dear benjamin ...I don t have the mass ... how can i do ?? Thx in advance
Dear Linda,
The good news about osteogenic differentiation is that it is not too tough to differentiate human MSCs into this lineage. I would definetely pay attention to the confluency of cells while differentiating them. With respect to formulation of media, I always added the osteogenic components like ascorbate, beta- glyderol phosphate and dexamethasone to the media righ before feeding the cells. So I would suggest making up a stock of your osteogenic components and then adding it to a smaller volume of media that you use for the cells. As Benjamin mentioned above, just use the volumetric equation to calculate. The molecular weights must be on the component vials themselves.
Good luck!
Aja
Dear Linda,
Regarding the calculations for osteogenic induction medium, this is how we follow.
First make stock solutions of Beta-GP (BGP) and Ascorbic Acid (AA)
BGP: 1M in PBS (216 milligram per mL of PBS)
AA : 5 milligram per mL of PBS
Now make small aliquots of this stock solution (like 1mL and 0.5 ml in small tubes and you can freeze them and store)
Now the final concentrations we usually use for the osteogenic induction is
BGP : 10mM & AA : 50 microgram per mL
Therefore just take the previously prepared stock aliquots and dilute it 100 times with media i.e, for 100 mL of media (theoretically for 98mL of media), add 1mL of stock BGP and 1ml of AA and for 50 mL (theoretically 49mL of media), add 0.5 mL of BGP and 0.5 mL of AA from the prepared and stored stocks.
Good luck.
Arun
hello aja ,
you wrote before :" So I would suggest making up a stock of your osteogenic components and then adding it to a smaller volume of media that you use for the cells. "
can i mix just all the osteogenic components togother to prepare the stock and then when i want to feed my cells i add a smal amount of osteogenic component to the media ? i mean i do it instead to prepare for example a stock of ascorbate , and a stock of beta glycerophosphate , hopw i can explain my questions well .
best regards
Hi Linda,
That is a good question.
Beta glycerol phosphate and Ascorbate are both water soluble. I would think it should be ok to prepare a stock with both the components added to it. Dexamethasone has to be dissolved in ethanol and a very small dose is added (10nM) to the media. I however have always had stocks of ascorbate, Beta glycerol phosphate and dexamethasone in individual vials. So, I can't tell you how a cocktail of all the factors as a stock would work from experience. Try it and let us know. Good luck!
Best,
Aja
Hi aja ,
I thinked to mix them , because i see that we can buy an osteogenic media and adipogenic media and chondrogenic media ready to use but there are very expensive because you know in the experiments we dont use one bottle and it is enough .
I will ask this question and i hope have an aswer and iwill let you know
Best regards
Linda
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