Cryoprotective Agent with 100 mm l-Glutamine (gCPA)
| Reagent | Source | mg/10 mL | mg/40 mL | Concentration |
|---|---|---|---|---|
| d+ Raffinose pentahydrate | Sigma-Aldrich R7630 | 1800 | 7200 | 18% |
| Dehydrated skim milk | BD Diagnostics, Difco 232100 | 300 | 1200 | 3% |
| l-Glutamine | Sigma-Aldrich G8540 | 146 | 584 | 100 mm |
| Cell culture–grade water | Invitrogen 15230–238 | 10 mL | 40 mL |
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1. Weigh l-glutamine, add it to the water, and vortex for 3 min.
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2. Place the tube into a 60°C water bath.
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3. Add the required amounts of raffinose and skim milk to the solution, and vortex for 3 min.
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4. Incubate for 90 min in a 60°C water bath. Vortex for 3 min every 30 min.
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5. Divide the solution into 1- or 1.5-mL aliquots and transfer into microfuge tubes.
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6. Centrifuge at 10,000g for 60 min. If the supernatant is not clear, centrifuge again until it becomes clear.
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7. Collect the clear top portion of the supernatant, leaving the undisturbed pellets behind.
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8. Filter the supernatant through a 0.22-µm filter. Aliquot into glass ampoules or microfuge tubes and seal with Parafilm.
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9. Store for up to 3 mo at room temperature.
