Immobilizing cells for live imaging

Scoop half of a sterile loop of cells (here we used Chlamydomonas reinhardtii and C. smithii ) from a lawn plate and vigorously agitate the loop along the inside of a 1.5 mL microcentrifuge tube containing 500 µL of the medium for the experiment (TAP medium or ddH₂O).

Leave the tubes with cells in a tube rack on the bench overnight with a light source illuminating them under a 12:12 hr light:dark cycle.

To make 1.25% low-melt agarose in TAP medium (which we use for Chlamydomonas cells), measure 3.75 mg of low-melt agarose and dissolve into 3 mL of TAP medium in a glass culture tube, then place it on a heat block set to 100 °C until the agarose dissolves.

Once dissolved, move the tubes to a heat block set at 45 °C and let the mixture acclimate for at least 20 min.

Prepare a working stock solution of vital dyes in an appropriate medium. For PKmito ORANGE, we used 1:500 in TAP medium. For staining two species of Chlamydomonas , we prepared 1 mL of dye to split (500 µl for each tube).

Pellet the cells by microcentrifugation 2000 × g for 2 min. Remove the supernatant and discard it.

Add 500 µL of the working stock of dye to each tube and mix by gently pipetting up and down, resuspending the pellet.

Transfer the cell/dye mixture to an opaque microcentrifuge tube (or wrap the tube with foil) to protect it from light and secure the cap with Parafilm.

Place the tubes on a rotator to incubate with the dye for 45 min (at room temperature).

After incubation, pellet and wash the cells twice with fresh TAP medium.

Draw a small wax circle (using a wax pencil) on a #1.5 glass coverslip (18 × 18 mm) for each sample (two separate coverslips for two tubes).

After removing the supernatant following the second wash, resuspend the pellet in 25 µL of the warmed (45 °C) agarose mixture.

Pipette 1 µL of the agarose/cell mixture within the wax circle on the coverslip.

Using forceps, carefully flip the coverslip onto a glass slide and seal the edges with VALAP.