Stereomicroscope

TGA and TGAC bioinks were bioprinted at 8 °C and 0.5 bar and
a speed of 4 mm/s. The formation of a filament or a drop after extrusion
was verified visually. Twelve filaments of 14 mm were bioprinted,
and images were acquired with a stereomicroscope (Leica). Their diameter
and length were calculated using ImageJ software (National Institutes
of Health). The filament diameter was determined at 30 different locations.
The spreading ratio was calculated by dividing the diameter of the
printed filament by the internal diameter of the needle.^{35 (link)} Filament fusion tests were performed with one
layer of the bioink. The scaffold showed the architecture described
in Figure 5A. The printability
and the diffusion rate were calculated as previously described.^{36 (link)} where A_t is the theoretical pore area, A_s the pore area of the scaffold, and L the perimeter
of the pore. The pore’s morphology was determined visually
by fabricating scaffolds with theoretical pores of 3 × 3 mm (area
after subtracting the thinness of the bioink, 0.51 mm of 6.2 mm²). The angle shape of filaments with an L shape was assessed
visually by bioprinting filaments as described in Figure 5A.

ORO staining was used to quantify lipids. (1) Fruit flies were dissected and the adipose tissue was exposed in phosphate-buffered saline (PBS). (2) PBS was removed and samples were fixed with 4% paraformaldehyde for 20 min. (3) Samples were washed three times with PBS for 10 min. (4) Oil Red O reagent was dropped on the samples, which were incubated for 1 h at room temperature. (5) The samples were washed three times with PBS for 10 min. (6) The samples were transferred to a slide with mounting solution. (7) Photos were taken with a Leica stereo microscope.

Overnight cultured C. albicans was diluted to 1 × 10⁷ CFU/mL with YNBB. Then, 0.1 mL of the diluted suspensions was inoculated to 24-well plates containing 0.5 mL CSE + 0.4 mL YNBB, or 0.5 mL boiled CSE + 0.4 mL YNBB in each well. The negative control contained 0.5 mL BHI and 0.4 mL YNBB in each well. The plates were incubated at 37 °C for 24 h anaerobically.
Crystal violet staining was performed to investigate the biofilm formation according to a previous study, with some modifications [37 (link)]. In brief, the supernatant in each well was removed. Then, the biofilms were gently rinsed three times with sterile phosphate buffer saline (PBS) and dried in the air. To fix the biofilms, 95% methanol was added. After 15 min, the methanol was removed and the biofilms were washed with PBS and dried in the air again. Subsequently, the biofilms were stained with 0.1% (w/v) crystal violet for 15 min, followed by removing the crystal violet solution, washing the biofilms with PBS and air drying. The images of biofilms stained by crystal violet were obtained by a stereomicroscope (Leica, Wetzlar, Germany). Then, 100% ethanol was added to each well to extract the crystal violet in biofilms. After 15 min, the ethanol was transferred to another 24-well plate and the optical density at 595 nm was detected using a spectrophotometer (Thermo, Waltham, MA, USA).

A nematode attraction test was performed as described by Wang et al. [49 (link)]. First, 23 g of pluronic F-127 powder (Sigma Aldrich, Brussels, Belgium) was added to 100 ml of sterile water and allowed to dissolve with stirring at 4 °C for 24 h. The rice roots from 2-week-old plants were drenched with 20 ml of 1.2 % biochar exudates or water. One day later, a 1-cm-long root tip was cut and placed into a 3.5-cm well in a 6-well culture plate containing 1 ml of pluronic gel and approximately 200 J2s. The nematodes in the vicinity of the root elongation zone were counted at 9 h post-inoculation (hpi), and photographs were taken under a Leica stereomicroscope with a DFC400 camera. The experiment was performed three times with 6 replicates each.

The binary expression vectors pGWB2-EgMAP20 (p35S:EgMAP20) and pEarleygate100-EgWDL3L (p35S:EgWDL3L) were constructed using the Gateway system, transformed into Agrobacterium C58 and used to transform wild-type Arabidopsis using the floral dip method [43 (link)]. T1 seed was harvested and EgMAP20 transgenic lines were selected on MS solid medium with kanamycin 50 μg/ml while EgWDL3L lines were screened using the herbicide BASTA. Positive seedlings were transplanted on soil. T2 generation seed of EgMAP20 and EgWDL3L and wild type seeds were plated on MS agar plates. After dark culture for 3 days, we observed hypocotyl cells of EgMAP20 seedlings under a stereomicroscope (Leica). After 12 days, EgMAP20 cotyledons were observed. Light and dark culture after 7 days, we observed EgWDL3L seedling hypocotyls, took pictures with a Nikon D300s camera and measured lengths using Image J.

500 cells mixed with 50% Matrigel were seeded in 24-well plates for 15 days. Medium containing the indicated concentrations of ciprofloxacin was exchanged every 3 days. Tumorspheres were imaged at day 15 using a Leica stereomicroscope, and quantification was conducted using ImageJ (ImageJ 1.53e; https://imagej.nih.gov/ij/index.html) software by measuring the number of tumorspheres per well based on the RFP reporter signal.