FAQ
- 用途別細胞培養
- MammoCult Medium (Human)で培養したMammosphereをカウントする方法を教えてください。
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マンモスフェアをカウントする際の操作方法については、以下の動画をご参照ください。
その他注意点はこちらです：
1. Harvest mammosphere or tumorsphere culture from the 6-well ultra low adherent dish and add them to a 14mL tube. The size of the tube will depend on the volume of the sample you are looking to evaluate.
2. Rinse each well that had previously contained culture with 2 mL of DMEM-F12 and combine with the collected suspension.
3. Centrifuge the suspension at ~350 x g for 5 minutes with the brake off.
4. Aspirate the supernatant carefully so you do not disturb the sphere pellet.
5. Gently resuspend the pellet in 500uL of complete MammoCult.
6. On the underside of a flat bottom 96-well plate draw a plus sign on a single well. This will help you divide the view of the spheres during counting. Note that if only one sample will be counted other wells of the plate can be reused at a later time provided they are kept covered.
7. Add 50uL of the sphere suspension to the plus sign-divided well of 96-well plate and observe under the microscope. We use calibrated eyepiece micrometers to determine the sphere size.
8. Now count spheres using a hand-tally counter. To ensure the accuracy of the count score at least 50 spheres in the well. If you have fewer spheres centrifuge the suspension again and resuspend it in a smaller volume. Keep in mind that due to potential cell and sphere aggregation this count provides just an estimate of the total number of spheres that formed.
