The key question this method addresses is, if a strain of interesting Chlamydomonas retains the ability for photo behaviors or not. This method is simple, quick and easy to test if a strain of interesting exhibits photo behaviors. This method can contribute to the photobiology and cell biology of ciliary motility.
Demonstrating the procedure will be Atsuko Isu, a lab technician from my laboratory. To begin, add two to three milliliters of cell suspension in a 3.5 centimeter Petri dish and place the dish on a light box. Shake the dish gently to uniformly distribute the cells and acquire a picture before illumination.
Illuminate the dish from one side with a green light-emitting diode or LED in a desktop dark room. Leave the dish for approximately five minutes before acquiring images. For phototaxis assay using cell culture droplets, place 25 microliters of cell suspension droplets directly onto a white plastic plate using a micro pipette.
Illuminate the droplets from one side with a green LED in a desktop dark room and leave them for three minutes before acquiring images. Place 30 microliters of cell suspension onto a glass slide and place an 18 by 18 millimeter cover slip with spacers on the side. Illuminate the samples from one side of the cover slip without a spacer with a green LED, and observe the cells under a darkfield microscope with a 10X objective lens under dim red light.
After light illumination, record the cell illumination for 20 seconds using a camera-equipped microscope. For the photoshock response assay, place 30 microliters of cell suspension onto a glass slide, and place a cover slip with spacers on top of the slide. Observe the cell under a microscope with dim red light.
Apply flash illumination using a camera flash while recording the cell movement. A representative example of negative phototaxis in Chlamydomonas reinhardtii after five minutes of side illumination is shown here. Most of the cells accumulated on the opposite side of the light source.
In the photoshock response assay, almost all cells exhibited the photoshock response. The cells swam backward for a short period and recovered forward swimming. To perform this procedure, use aridimigudolphase cells and expose them to the red light before the assay.
If not exposed to the red light, cells will become insensitive to the light. This technique isolates various mutants with abnormal phototaxis of photoshock responses. In addition, various intracellular and extracellular factors that influence photo behaviors have been identified.
