Methylene blue

The cells were seeded in 6-well plate as 90% confluence, after overnight attachment the media was changed to starvation media with or without βHb 3 or 10mM for 7 days. The cells to be stained were washed with PBSx1 and methanol with methylene blue [0.5% methylene blue (Cat#M4159, Sigma-Aldrich) dissolved in 50% methanol (Cat#S93301, Thermo-Fisher) and complete volume with ddH₂O] was added and incubated for 4 h at room temperature. The plates were washed in water and imaged after air-drying.

All leaf segments were washed thoroughly with tap water to remove the sticky glue from the surface before staining. ruthenium red is used on plant materials for staining pectin, mucilage, and gum [37 (link)]. Leaf segments were incubated in a 0.01% (w/v) ruthenium red (Sigma-Aldrich, St. Louis, MO, USA) solution for 10 min to stain pectic acid. Moreover, as methylene blue is not able to penetrate intact cuticles [2 ], leaf segments were stained with a 0.1% (w/v) solution of methylene blue (Sigma-Aldrich, St. Louis, MO, USA) for 10 min to detect cuticle pores. Leaf segments were washed twice to remove excess dye before being observed under a dissection microscope (Nikon, SMZ745T, Tokyo, Japan).

To screen strains expressing mutant alleles of CBP1, J774.1 cells were seeded (3.75 x 10⁴ cells per well of a 24-well plate) and infected as described above in triplicate wells per time point. Three independent transformants per alanine mutant were tested. Each day for 4 days after infection, macrophage monolayers were washed once with PBS, fixed and stained with methylene blue staining solution (0.2% methylene blue [Sigma Aldrich], 20% ethanol) at room temperature for 15 min, washed 3 times with PBS, and then imaged.
To quantify macrophage lysis, BMDMs were seeded (7.5 x 10⁴ cells per well of a 48-well plate) and infected as described above. At the indicated time points, the amount of LDH in the supernatant was measured as described previously [65 (link)]. BMDM lysis is calculated as the percentage of total LDH from uninfected macrophages lysed in 1% Triton-X at the time of infection. Due to continued replication of BMDMs over the course of the experiment, the total LDH at later time points is greater than the total LDH from the initial time point, resulting in an apparent lysis that is greater than 100%.

This study compared three different photosensitizers: a phenothiazine derivative (methylene blue); a chlorophyll derivative (chlorin-e6); and a turmeric curcuminoid (curcumin).
methylene blue was purchased from Sigma (Sigma-Aldrich Co. LLC, St. Louis, MO, USA). Chlorin-e6 was synthesized as described by Uliana and co-workers^{53 (link)}, and curcumin was synthesized as described by Wichitnithad and co-workers^{54 (link)}.

The water stimulus was three droplets of distilled water with 0.1% Methylene Blue (MilliporeSigma). The beads stimulus was 4–5 of red polystyrene beads (4.7mm in diameter). The food extract was made by suspending 0.1 g of fine ground Tropical XL Color Granules with Natural Color Enhancer (Tetra U.S., Blacksburg, VA) in 2 mL of distilled water mixed with 0.5 mL of 0.5% Methylene Blue (MilliporeSigma) and filtered with a 0.45 μm syringe filter. The food extract was made fresh for each experiment and three drops were added as the stimulus. The agar-solidified food was comprised of 1.0 g of fine ground Tropical XL Color Granules with Natural Color Enhancer (red colored granules) suspended with 5 mL of 1% agar (MilliporeSigma) in the fish conditioned water (pH 6.8–7.0, conductivity ~700 μS), then poured into 6-cm dishes to solidify. Once solidified, a razor blade sterilized with 70% ethanol was used to cut the agar food into 5 × 5 mm squares and 3–4 pieces were given per stimulus. Sinking of red plastic beads was approximately the same as the red agar food, mimicking red agar food movement.