Proliferation assay of mouse pluripotent ES cells

Summary

This experiment is based on the "Guide to Cellular Experiments", translated by Huang Peitang et al.

Operation method

Culture of pluripotent ES cells

Materials and Instruments

PBS Trypsin solution
MEF trophoblast plate Pasteur pipette ES growth medium

Move

1. Prepare the following reagents and materials:

60 mm MEF nourishment plates (inoculation should not exceed 7 days)

Sterile Pasteur pipettes with cotton plugs

^Ca2+ and ^Mg2+ free PBS

ES Growth Medium

Trypsin solution

2. PBS rinse ES cells, Pasteur pipette with trypsin solution to cover the bottom of the dish (2 ml/p60)

3. Incubate at 37℃ for about 5 minutes until the cells are detached.

4. Gently blow the trypsin-treated cells with a Pasteur pipette 4-6 times to disperse the ES cells. Dispersed cells, including single cells and small cell clusters, are transferred to a 15 ml centrifuge tube containing 2 ml of pre-warmed (37°C) ES growth medium.

5. Collect the cells by centrifugation. Aspirate off the supernatant, leaving approximately twice the volume of cell sediment. Flick the tube to suspend the cells. Add 10 ml of ES Growth Medium and aspirate until the suspension is homogenized.

6. Count the cells. Since ES cells are smaller and more rounded than MEF cells, they can be easily distinguished.

7. Inoculate ES cells into 60 mm MEF trophoblast plate. Aspirate off the MEF trophoblast medium and add ES cells suspended in 5 ml of ES growth medium.

8. Add 5 ml of ES growth medium with a daily fluid change.

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