Collect medium from homologous or heterologous cell lines in late logarithmic growth, filter, and dilute with fresh medium as needed.

Operation method

Scheme 14.2 Preparation of Conditioned Media

Principle

Collect medium from homologous or heterologous cell lines in late logarithmic growth, filter, and dilute with fresh medium as needed.

Materials and Instruments

Conditioned Cells
Culture medium for cloning Sterilization filters

Move

1. Conditioned cells (Conditioned cells: same cell, other cell line (e.g., 3T 3 cells), or mouse embryonic fibroblasts) were grown to 50 % confluence.

2. Change the culture medium and continue incubation for 48 h.

3. Collect the medium.

4. Centrifuge at 1000 g for 10 min.

5. Filter through 0.45 μm sterile filters (decontamination filters: 0.45 μm or 0.22 μm, filter bottles) (the medium can be pre-filtered through 5 μm and 1.2 μm filters first).

6. Store the conditioned medium (cloning medium: Ham's F12 with 10 % FBS or a medium suitable for the cells to be cloned) at -20°C.

7. Thaw before use and add to the cloning medium in the following ratio: 1 part conditioned medium to 2 parts cloning medium.

Common Problems

Centrifugation, freezing, thawing, and filtration are all steps that help avoid carrying cells from the original culture flask. This problem is a little lighter if the cells in the clonal culture are the same cells as the conditioned cells. But using a different cell line or primary mouse fibroblasts as conditioned cells may be more conducive to clonal culture. If a cell line is established in this way, the nature of the cells must be characterized (see Scheme 16. 10, Scheme 16. 11, Scheme 16. 12) to exclude cross-contamination of the cells due to the conditioned medium.

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