Dissolving nucleosides for use in cell culture studies
Dr. Linda L. Wotring and Dr. Dean S. Wise
Try PBS (Physiological Buffered Saline) first.
1. Calculate and weigh out 1.1 times the amount (approx. 1 mg) of nucleoside that will be required to make 100 mL of a 10-4 M solution.
2. Transfer the weighed-out nucleoside to a tube and add 5 mL of PBS. Shake gently to dissolve, warm slightly if necessary.
3. If the nucleoside dissolves, filter-sterilize the PBS solution (0.22 micron or smaller pores).
4. Place 96 mL of culture medium in a flask and add 4.5 mL of the sterile PBS solution.
5. Make serial dilutions to obtain lower concentrations, if desired.
If the nucleoside does not dissolve in PBS, then try DMSO.
1. Dissolve the same amount of nucleoside in 0.1 mL of DMSO, warm slightly if necessary and cool to room temperature.
2. In a beaker (about 400 mL in size) on a magnetic stirrer, place 110 mL of culture medium. It is easier to re-sterilize if the medium is without serum, but the nucleoside may dissolve better with serum present. Stir to near vortex. Do not allow any vortex to form, as this will cause precipitation of the nucleoside.
3. Using a Pasteur pipette, add as much of the DMSO solution as possible to the medium in a dropwise fashion. Add the drops at one half the radius of the beaker, allowing time for complete stirring between drops. Do not worry about any residual DMSO solution in the tube or the pipette.
4. If no visible precipitate has formed, filter-sterilize the solution. A filter (0.22 micron or smaller) holder with a syringe adapter works well with the largest feasible syringe, usually 50 mL. Do not worry about any compound left on the filter.
5. Run a DMSO (no nucleoside) control with the experiment.
If both of these methods fail, try using the DMSO procedure with 1/10th the concentration of nucleoside, and making 10-5 M as the most concentrated solution in culture medium. However, there may still a visible precipitate when the DMSO solution is introduced into the medium. If a still smaller amount of nucleoside is used, you may not be able to see the precipitate if it forms.
NOTE: The concentration of the nucleoside that cells are actually exposed to may be less than 10-4 M, particularly with the DMSO method.