Axiovision a1

Cell density, cell volume and cell circularity were determined on a Nageotte slide after cell immobilization (500:50 v/v) with a 4% solution of paraformaldehyde (PF), (ACROS Organics, Illkirch, France, >96%) in saline phosphate buffer. The treatment did not affect the morphology or the colour of the microalgae [41 (link)]. Sixty microscopy bright fields (AxioVision A1, Zeiss, Iena, Germany; magnification: ×400, picture resolution: 0.25 µm px⁻¹) were pictured randomly. The colour pictures (16 bit) were transformed into B/W ones (8 bit) using the Image J software (https://imagej.nih.gov/ij/, accessed date: 9 October 2021, National Institutes of Health), which were then subjected to particle analysis using a default Image J routine. Briefly, cell debris and other small particles were first automatically removed from the analysis by setting the “cell area” parameter to the range 20–400 µm² and by manual sorting. A minimum of 1600 cells were counted, limiting the standard error of cell concentration estimation to 5% [42 (link)]. Cell density was calculated as the number of cells (N_cells) in the corresponding volume of the image sample (V_image= 4.75 × 10⁻² µL) multiplied by the number of images (N_images), adjusted with the PF dilution coefficient (dil_PF) as shown in Equation (4):

DD-Csy4 expression was controlled by adding Shield1 into the culture medium. To analyze the expression of HA-tagged DD-Csy4, whole cell extracts were subjected to SDS-PAGE, and the protein level was determined by Western blotting as described previously [72 (link)] using anti-HA (Roche; 3F10; 1:5,000) and anti-α-TUBULIN (Abcam; ab7291; 1:10,000). Fluorescent protein expression was observed under AxioVision A1 (Zeiss) and quantified using a Gallios flow cytometer (Beckman Coulter). Mean fluorescence intensity was calculated using Kaluza software (Beckman Coulter).