I have made a 6% solution by mixing a polysaccharide in deionized water. Now, I want to treat cells in a 12-well plate with this solution.
The problem that now arises is; once I add the solution, it will become diluted due to the cell medium, and the medium (DMEM 10% bovine serum) will become diluted as well. How may I solve this? We wish to treat the cells with 6%, 3% and 1% solution. Any ideas? Formulas? The well should have a total volume of 1-2 mL.
Thank you for any helpful thoughts.
Hans,
when adding such large amounts of water to cells, you'll induce osmotic stress in your cells which might mask the effect your looking for.
Standard way is to dilute the drug to be tested in the same medium as your cells are in, so the only parameter that is changing is the drug concentration. If possible, make appropriate dilutions, so that an equal volume (with different drug concentrations) is added to the cells, in order to exclude effects of nutrient supply.
When your cells are adherent, you also simply could change the medium instead of adding it (which also avoids effects of uneven mixing).
Hello Hans Erling Skallevold
I suggest you make a higher percentage of the stock solution of polysaccharide because it is not possible to have a 6% stock polysaccharide solution when you need 6% as the working solution. You preferably make the stock solution in plain medium (DMEM without FBS).
So, I suggest you do the following:
Suppose you need 1 ml of culture medium per well (as you have mentioned above) in the 12-well plate, and for 6% of polysaccharide solution (final concentration) to be added in the well you need to do the following.
For 6% polysaccharide solution to be present in the well
If you make :
10% stock solution of polysaccharide = 600 ul of stock (10%) + 400ul of culture medium.
12% stock solution of polysaccharide= 500 ul of stock (12%) + 500ul of culture medium
15% stock solution of polysaccharide= 400 ul of stock (15%) + 600ul of culture medium.
If you notice the higher the percentage of stock solution, the lesser is the volume of the stock solution to be added finally in the well. So, I would prefer you add 100ul of the 60% stock polysaccharide solution and 900ul of culture medium to make the volume to 1 ml. Make the dilutions in sterile 1.5ml appendof tubes, mix well by pipetting and add to the well. The more the volume of complete medium the better will be the supply of nutrients to the cells.
Similarly, for 3% and 1% you need to add as follows:
3% polysaccharide working solution = 50ul of 60% stock polysaccharide solution + 950ul of (DMEM + 10% FBS).
1% polysaccharide working solution =16.67ul of 60% stock polysaccharide solution + 983.33ul of (DMEM + 10% FBS).
You can use the formula N1V1=N2V2
Where N1 = percentage of the stock polysaccharide solution (60%)( which you need to prepare instead of 6%).
V1 = volume of the stock polysaccharide solution to be added (X ul).
N2= percentage of the working solution in the well (6% or 3% or 1%).
V2= total volume in the well (1ml).
So, it is better to make higher percentage of the polysaccharide stock solution.
I hope this is helpful.
Good Luck.
Hans,
I agree with @wolfgang addition of deionised water is large amounts would kill your cells due to the osmotic stress. The best approach is to dissolve the polysaccharide in the media itself weight/volume. However if this is not feasible my idea would be make a highly concentrated stock solution of the polysaccharide. Since you did not mention the polysaccharide of working with I really cannot know what is the highest concentration you could achieve. But suppose you make a 50% stock solution of the polysaccharide. Now keeping your cell well volume of media added constant apply the formula V1S1 = V2S2. Suppose your media volume to be added to each cell is 3 ml..so the formula would be 3x6=V2x50
Therefore it means you need to add 0.36 ml of your stock solution to 2.64 ml of media to achieve a 6% strength. The same formula could be applied to 3% and 1%.
Hope this helps
Arup
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