Inoculation of homologous or heterologous cells-such as those from mouse embryos. Pending clonal culture of the cell under test, medium density, irradiated with radiation or drugged to render them incapable of proliferation.

Operation method

Program 14.3 Preparation of feeding layers

Principle

Inoculation of homologous or heterologous cells-such as those from mouse embryos. Pending clonal culture of the cell under test, medium density, irradiated with radiation or drugged to render them incapable of proliferation.

Materials and Instruments

Second-generation 13-day-old mouse embryonic fibroblasts mitomycin C
Medium for cloning culture X-ray or 6GCo source

Move

1. Digest primary cultured embryonic fibroblasts with trypsin (see Protocol 12.6, Protocol 13.2) and replant into culture flasks at ¹⁰⁵ cells per ml.

2. Block further division by one of the following methods:



(a) treatment by radiation

i) In the original culture flask, cells are exposed to 60 Gy by irradiating the cells with an X-ray machine or a γ-ray source such as ^60Co (X-rays or ^60Co source that can release 30 Gy in 30 min or less (can be substituted for mitomycin C)).

ii) After digestion of the cell suspension, the cells are exposed to 60 Gy by irradiating the cells with an X-ray machine or a γ-ray source such as ^60Co. The cells are then inoculated at 1 x ¹⁰⁵ ml or the irradiated cells are stored at 4°C for up to 5 days.

(b) Treatment by mitomycin C (mitomycin C, 100 μg/ml stock solution, dissolved in HBSS or serum-free medium)

i) Add mitomycin at 0.25 μg/ml to near-confluent cells overnight at 37°C (~18 h) [ Macpherson and Bryden, 1971 ] and replace the medium.

ii) Trypsin-digested suspensions of 1 × ¹⁰⁷ ml were spiked with mitomycin C at a final concentration of 20 μg/ml (equivalent to 2 ^μg/106 cells), incubated at 37°C for 1 h, washed by centrifugation (4 × 10 ml) to remove mitomycin, and then inoculated at 1 × ¹⁰⁵ /ml [Stanley, 2002] or stored at 4°C for up to 5 days.

3. After 24-72 h of further incubation, trypsin digest and inoculate in new medium. Or directly inoculate the preserved cells. Inoculate at a concentration of ^5×104 cells/m l ( ^104cells/cm2 ).

4. Continue incubation for 24-48 h, and then inoculate with clonally cultured cells.

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