Sexual pathway (binding) induction experiments

Summary

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

Operation method

Sexual pathway (binding) induction

Materials and Instruments

Tetrahymena
Tris
Culture medium Culture flasks

Move

Culture Preparation

1. For each mating type, inoculate 5 ml of recently transferred short-storage Tetrahymena spp. in a 1 L culture flask containing 100 ml of medium and cover the flask with an aluminum foil or cotton wool stopper.

2. 30°C Incubate to late logarithmic phase, usually this culture is stationary. Under these conditions, the cells will reach the desired density within 24 hours.

Starve the cells in 10 mmol/L Tris to initiate starvation.

3. Wash the precipitated cells by centrifugation at low speed in 10 mmol/L Tris at pH 7.4, discard the supernatant, and gently suspend the cells in Tris. Centrifuge at low speed and discard the supernatant.

4. Resuspend each mating cell in 10 mmol/L Tris to a final concentration of ^2X105~2. ^5X105 cells/ml. aliquot 100 ml into 1 L culture flask. Make sure to keep the mating type isolated.

5. Cover the culture flasks with aluminum foil or cotton wool stoppers and incubate overnight at 30°C with no shaking.

Cell co-stimulation and pair formation

6. Slightly synthesize cells of the same mating type and assay the concentration.

7. Mix equal numbers of cells of opposite mating type and aliquot 50~80 ml in each 1 L culture flask and cover with aluminum foil or cotton wool stopper.

8. The cells should be allowed to stand at 30°C until the desired growth period is reached.

9. At the desired growth period, harvest mating cells by centrifugation at 1500-2000 g for 5 minutes. The morphology and number of nuclei are convenient characteristics for determining the growth period.

10. If isolated nuclei are prepared, resuspend the cells in medium at a concentration of ^{2X106 cells/ml} and store frozen at -20°C or use directly.

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