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ICD-10-CM code: C41, malignant neoplasm of bone and articular cartilage metastatic Ewing sarcoma

Propagenix Conditioned Medium (Propagenix cat# 256-100) supplemented with 9.0 ng/mL cholera toxin (Sigma Aldrich C8052).

Prepare media according to the manufacturer’s instructions

Important: Pool both the adherent and suspension cell populations when passaging.

1. Passage cells when the culture has reached approximately 70% to 80% confluence.
2. Warm TrypLE (Thermofisher # 12605010) and complete growth media to room temperature.
3. For each flask, carefully aspirate the spent media without disturbing the monolayer.
4. Rinse the cell layer one time with 3 to 5 mL D-PBS (ATCC 30-2200) to remove residual medium.
5. Add room temperature TrypLE (1 to 2 mL for every 25 cm²) to each flask.
6. Gently rock each flask to ensure complete coverage of the TrypLE solution over the cells, and then aspirate the excess fluid off of the monolayer.
7. Observe the cells under the microscope.
8. When the majority of cells appear to have detached (typically 2-5 minutes), quickly add an equal volume of the complete growth medium to each flask.
9. Transfer the dissociated cells to a sterile centrifuge tube.
10. Centrifuge the cells at 200 x g for 5 minutes.
11. Aspirate neutralized dissociation solution from the cell pellet and resuspend the cells in 2 to 8 mL fresh, pre-warmed, complete growth medium.
12. Count the cells and seed new culture flasks at a density of 2 to 5 x 10⁴ viable cells per cm². Prior to seeding, aspirate the coating and discard the coating laminin solution from the vessel.
13. Place newly seeded flasks in a 37°C, 5% CO₂ incubator for at least 24 to 48 hours before processing the cells further.
14. Perform a complete medium change every 3-4 days or as needed.