TITLE: Choanoflagellate Ciliogenesis Live Imaging
AUTHORS: Maxwell C Coyle

[DESCRIPTION]
This protocol removes the cilia/flagella from choanoflagellate cells and sets up the cells for live imaging of ciliogenesis. It has been developed for the species Salpingoeca rosetta, but may be portable into other choanoflagellate species. Cells begin to re-generate their cilia/flagella right after removal. The idea of using a cold shock with glycerol for ciliary removal came from Brokaw et al 1960 (doi: 10.1016/0014-4827(60)90027-6).

[STEPS]
SECTION: Concentrate cells and remove cilia
1. Grow choanoflagellate cells (Salpingoeca rosetta fed with Echinicola pacifica, ATCC PRA-390) in High Nutrient Media to a density of 1-2 x 106 cells/ml. Grow at 22 ºC, 60% humidity

We grow 30 ml of culture in 75 cm2 vented flask. Typically, inoculating this flask with a choanoflagellate cell density of 8,000 cells/ml 48 hours before ciliogenesis works well.
SECTION: Concentrate cells and remove cilia
2. Count cells by haemacytomer or automated cell counter*. Shake culture flask vigorously to homogenize cell populatin and thene mix 99 µl of cell culture with 1 µl of 16% paraformaldehyde to fix cells for counting. Typically 10 µl of fixed cells can be loaded into a haemocytometer or automateed cell counting slide.

*We use LUNA-FL Automated Fluorescence Cell Counter (Logos Biosystems L20001)
SECTION: Concentrate cells and remove cilia
3. Aliquot and pellet 6 x 106 cells 2000 x g, 10 min 

SECTION: Concentrate cells and remove cilia
4. Corona treat a fluorodish 5-10 seconds  
 

SECTION: Concentrate cells and remove cilia
5. Rinse fluorodish for 5 seconds with 1 ml of 0.1 mg/ml poly-D-lysine, followed by 3x washes with 1 ml water. Dry by air or by capillary action of kimwipe, being careful to minimize contact with surface of imaging dish.

 
SECTION: Concentrate cells and remove cilia
6. Rinse a coverslip (circular - 22mm diameter) in 70% EtOH followed by water and then lay on kimwipe to dry. Easiest to hold coverslip by forceps and dunk into 50 ml conical tubes with the ethanol or water.
SECTION: Concentrate cells and remove cilia
7. When cells are done pelleting, remove supernatant and resuspend cell pellet in 800 µl of AKSW and transfer to 1.5 ml eppendorf tube.
SECTION: Concentrate cells and remove cilia
8. Add 200 µl of 50% glycerol (final concentration: 10% glycerol) and mix by pipetting
SECTION: Concentrate cells and remove cilia
9. Add cells to a second Fluorodish (i.e. one not treated with poly-D-lysine) and incubate-20 °C 

Standard laboratory freezer is fine, but depending on exact temperature of your freezer or where in the freezer you place the cells, you may need to adjust the timing.
SECTION: Set up cells for live imaging of ciliogenesis
10. Transfer cells to 1.5 ml eppendorf tube and pellet 4200 x g, 8 min 

SECTION: Set up cells for live imaging of ciliogenesis
11. Remove supernatant and resuspend cells in 25 µl of AKSW
SECTION: Set up cells for live imaging of ciliogenesis
12. Transfer cells to Fluorodish coated with poly-D-lysine and lay clean coverslip slowly on top using forceps
SECTION: Set up cells for live imaging of ciliogenesis
13. Mount dish on microscope* and find focus. Let cells settle for 1 minute.

*We use a Zeiss Axio Observer.Z1/7 widefield with a 100x NA 1.40 Plan-Apochromatic oil immersion objective and a Hamamatsu Orca-Flash 4.0 LT CMOS digital camera
SECTION: Set up cells for live imaging of ciliogenesis
14. Float coverslip off of cells by adding AKSW around the side of the coverslip drop by drop with a plastic transfer pipette. If you don't do this, the cells will eventually suffocate.
SECTION: Set up cells for live imaging of ciliogenesis
15. Image!

On our system we use a short (5 ms exposure) with high light intensity (12.2 V) and a DIC condenser to get the best imaging of ciliogenesis. We use Zeiss Definite Focus and take a 10 µm z-stack with 1 µm between slices every 30 seconds for one hour.