I am planning to perform C13 MFA, and I was wondering how I can weigh the labeled glucose and add it to the media while maintaining sterility. I didn't find any protocol specifically explain this part.
Performing C13 metabolic flux analysis (MFA) typically involves working under sterile conditions to ensure the accuracy of your results. To weigh and add labeled glucose to the media while maintaining sterility, you can follow these steps:
1. Prepare a sterile laminar flow hood or biosafety cabinet to work in.
2. Wear appropriate personal protective equipment (lab coat, gloves, and possibly a face mask) to prevent contamination.
3. Weigh the required amount of labeled C13 glucose on a sterile analytical balance within the sterile environment.
4. Use a sterile spatula or scoop to transfer the weighed C13 glucose into a sterile container. You can use a sterile beaker or flask for this purpose.
5. Prepare your cell culture media separately in a sterile flask or container.
6. In a laminar flow hood, under sterile conditions, aseptically transfer the labeled glucose from the sterile container into the media using a sterile pipette or syringe. Be cautious not to introduce any contaminants during this step.
7. Mix the media thoroughly to ensure even distribution of the labeled glucose.
8. Once the media is prepared with the labeled glucose, it's essential to maintain sterility throughout the entire experiment. Ensure that all equipment, containers, and surfaces that come into contact with your culture remain sterile.
Remember to consult specific guidelines or protocols for C13 MFA in your field or from your institution to ensure you follow the recommended procedures for your experiment. Additionally, maintaining good aseptic techniques is critical to the success of your study.
Assuming that the labeled glucose powder is not sterile, you have 3 options.
1. Prepare a concentrated stock solution of the labeled glucose and pass it through a sterile syringe filter into a sterile container.
2. Prepare the medium from powder, including the labeled glucose powder, and then sterilize it in the usual way.
3. Add the non-sterile labeled glucose as powder or stock solution directly to sterile medium, then re-sterilize the medium by membrane filtration.
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