Evans blue dye solution

Lung vascular leak was measured in each experimental group using (1) Evans blue assay in lung tissue, (2) lung water content, and (3) BALF protein concentrations. The Evans blue dye assay was performed as described previously [19 ]. Briefly, the mice were administered 1% Evans blue dye solution (Sigma, St. Louis, MO, USA) in saline via tail vein injection. After 40 min, the mice were sacrificed and perfused via the heart, and the lung tissues were collected. The lung weights were measured and placed in 1 ml of formamide (Avantor, Center Valley, PA, USA) at 60 °C for 24 h to extract Evans blue dye. The samples were centrifuged at 2000 rpm for 10 min, and the supernatants were collected. The concentrations of Evans blue dye in the supernatants were quantified by measuring absorbance at 620 nm and calculated from a standard curve by a plate reader.
For lung water content, the left lung was harvested and weighed to measure a wet weight in each group. The wet lung was then dried in an oven at 60 °C for 48 h and re-weighed as dry weight. The lung water content was calculated as the ratio of wet weight to dry weight. Protein levels in the BALF supernatant were determined by DC protein assay (Bio-Rad, Hercules, CA, USA).

Lung vascular permeability was measured in each experiment. The Evans blue assay was used to test vascular permeability in lung tissue and was performed as described previously (Radu and Chernoff, 2013 (link); Pati et al., 2018 (link); Zhou et al., 2019 (link)). Briefly, the mice received an intravenous injection of 1% Evans blue dye solution (Sigma, St. Louis, MO, USA). After 1 h, the mice were sacrificed and perfused via the right ventricle with 4°C PBS for 10 min to introduce the dye intravascularly, and the lung tissues were collected. The lungs were placed in 1 ml of formamide (Avantor, Center Valley, PA, USA) at 60°C for 24 h to extract the Evans blue dye. The lung tissues were then centrifuged at 2,000 rpm for 10 min, and the supernatants were collected. The concentrations of Evans blue dye in the supernatant were measured at an absorbance of 620 nm. For lung water content, the left lung was harvested and weighed to determine the wet weight in each group. The lungs were then dried to a constant weight at 60°C for 48 h and were weighed to determine the dry weight. The lung water content was calculated as the ratio of wet weight to dry weight.

Evans blue dye solution (0.02%, Sigma-Aldrich) was mixed with an equal volume of sorghum protoplasts in W5 solution and the mixture was incubated at 25 ℃ for 10 min. The numbers of live (unstained) and dead (stained) protoplasts were determined on a hemocytometer under a light microscope. Protoplast viability was calculated as the number of unstained protoplasts / total number of protoplasts.