This method can help to understand which factors affect cilia motility. The main advantage of this technique is that the phenomena that occur only temporarily in vivo, such inversion of calcium ion concentrations in cilia can be continuously observed in vitro. This method contributes specifically to the research field of motile cilia.
It would be difficult for beginners to perform the demembranation step. Hence, it is highly recommended to carry out the steps gently but precisely. Begin by centrifuging approximately 10 milliliters of Chlamydomonas reinhardtii culture at 1, 000 RCF at 20 degrees Celsius for three minutes.
Decant the supernatant and aspirate the remnant by a Pasteur pipette. Resuspend the precipitates in approximately five milliliters of washing buffer. Centrifuge cells in the washing buffer at 1, 000 RCF for three minutes at 20 degrees Celsius.
Discard the supernatant carefully with a pipette. Add approximately 0.5 milliliters of demembranation buffer to the cell pellet. Shake the tube gently to roughly suspend the cells in the buffer, then put the tube on ice.
Suspend the remaining cell pellet gently with a pipette and place the tube on the ice again. Take 5 to 10 microliters of the cell model, dilute tenfold with the dilution buffer, and observe under the microscope to confirm all the cell models are demembranated and not swimming. In a 0.5-milliliter tube, mix 80 microliters of reactivation solution, 10 microliters of ATP solution, and 10 microliters of cell models by tapping the tube.
Put approximately 30 microliters of the mixed solution onto a glass slide and gently place a cover slip on it with spacers to avoid a mechanical shock to the cell model. Observe the reactivated cell models under a microscope. After demembranation of the C.reinhardtii culture, all cell models became immotile and the demembranated cilia remained attached to the cell body, confirming that the immotility of the cell models was not caused by de-ciliation.
After mixing the cell models with the reactivation buffer in ATP, more than 50%of cell models became motile. Even without the ATP regeneration system, the reactivated cells continued to move for approximately 90 minutes with a final ATP concentration of one millimolar before gradually stopping. The demembranation step is critical.
All the cells should stop swimming, but they should not be mixed so strongly that de-ciliation occurs. Researchers can modify this protocol to include reagents such as calcium ions or cyclic AMP, or perform experiments with a mutant lacking a particular protein to identify their biological role.
