Cryoprotective Medium (CPM)
| Reagent | Source | Quantity | Final concentration |
|---|---|---|---|
| d+ raffinose pentahydrate | Sigma-Aldrich R7630 | 45 g | 18% |
| Dehydrated skim milk | BD Diagnostics 232100 | 7.5 g | 3% |
| Monothioglycerol (MTG; FW = 108.16; ρ = 1.295 g/mL) | Sigma-Aldrich M6145 | 10 µL | 447 µm |
| Cell culture-grade water | Invitrogen 15230-238 | to 250 mL |
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1. Warm 150 mL of distilled culture-grade water covered in a microwave for 25–40 sec.Heating the water makes it easier to get the concentrated raffinose into solution.
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2. Place the container of warm water on a stir plate with a stir bar. Add 45 g of raffinose to the water. Stir until it is completely dissolved.
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3. Slowly add 7.5 g of dehydrated skim milk. Stir until the skim milk has gone into solution.
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4. Add 10 µL of monothioglycerol (MTG) into the solution, and mix thoroughly.
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MTG is viscous, making careful pipetting essential.
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5. Place the solution in a volumetric flask, and bring to a volume of 250 mL with culture-grade water. Mix thoroughly.
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6. Centrifuge at 13,000g for 15–30 min at room temperature to clarify the solution. If the supernatant is not clear, centrifuge again.
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7. Immediately after centrifugation, slowly pour the CPM into a 0.22-µm cellulose acetate filter unit, being careful not to dislodge the pellet when decanting. Two filter units may be required to complete filtration of the entire 250 mL.
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8. Test osmolarity; it should be 470–490 mOsm.
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9. Aliquot as required and store for up to 3 mo at −80°C. Thaw at 37°C before use and mix to redissolve the raffinose.
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Do not refreeze. Solutions containing MTG should not be kept at 4°C.
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