Sodium hypochlorite

The following reagents (with their sources indicated in parentheses) were used: acetonitrile and methanol (LC-MS grade, J.T. Baker), formic acid (EMD), dithiothreitol (Bio-Rad), N-ethylmaleimide (Pierce), sodium hypochlorite (Fisher Scientific), L-cysteine-¹³C₃¹⁵N, N-acetyl cysteine-¹³C₃¹⁵N, cystine-¹³C₆¹⁵N₂, homocysteine-3,3,4,4-d₄, and caffeine-¹³C₃ (Cambridge Isotope Laboratories). L-cysteine-glutathione disulfide (Cayman). All other reagents were obtained from Sigma-Aldrich.Table 2

Composition and concentration of analytes in the QC standard (all thiols were blocked with NEM before adding to the mixture).

Name	Abbreviation	Conc (µM)
L-cysteine	Cys	8.0
Glutathione	GSH	2.2
N-acetyl cysteine	NAC	10.0
L-homocysteine	Hcy	3.0
L-cysteine-L-glycine	CG	3.0
γ-L-glutamyl-L-cysteine	γ-EC	1.1
Cystine	Cystine	15.4
Glutathione disulfide	GSSG	2.0
N-acetyl cysteine disulfide	NACss	8.0
L-cysteine-L-glycine disulfide	CGss	2.6
L-cysteine-glutathione disulfide	GSH-ss-Cys	6.0
L-cysteine-homocysteine disulfide	Hcy-ss-Cys	3.2
L-cysteine-N-acetyle cysteine disulfide	NAC-ss-cys	5.4
L-cysteine-CysGly disulfide	CG-ss-Cys	6.9
L-cysteine-γGluCys disulfide	γ-EC-ss-Cys	0.8
Caffeine	Caff	4.2

For all experiments, zebrafish embryos were initially derived germ-free using previously described gnotobiotic procedures with slight modification [75 (link)]. Briefly, fertilized eggs from adult mating pairs were harvested and incubated in sterile embryo media (EM) containing ampicillin (100 μg/mL), gentamicin (10 μg/mL), amphotericin B (250 ng/mL; MP Biomedicals, Santa Ana, CA), tetracycline (1 μg/mL; Sigma-Aldrich, St. Louis, MO), and chloramphenicol (1 μg/mL; Amresco, Solon, OH) for approximately 6 hours. Embryos were then washed in EM containing 0.1% polyvinylpyrrolidone-iodine (Syndel, Ferndale, WA) followed by EM containing 0.003% sodium hypochlorite (Fisher Scientific, Hampton, NH). Surface sterilized embryos were distributed into T25 tissue culture flasks (TPP, Trasadingen, Switzerland) containing 15 mL sterile EM at a density of 1 embryo/mL and kept in a temperature-controlled room at 28°C to 30°C with a 14 hours/ 10 hours light/dark cycle. The germ-free status of larval zebrafish was assessed before every experiment by visually inspecting flask water for microbial contaminants using an inverted microscope. Culture-based assessment of germ-free status was done as needed by plating 100 μL flask water on rich media (e.g., tryptic soy agar). Embryos were sustained on yolk-derived nutrients and not fed prior to or during any experiments.

This involved about 100 genera of field-plants covering cereal, pulse, oilseed, fruit, vegetable, ornamental, medicinal, tuber and forage crops, grasses, weeds, and forest species (Table 1). The list included herbs, shrubs, climbers, and trees across annual and perennial species covering dicots and monocots mostly from India (Bengaluru, Karnataka State, or parts of neighboring Kerala and Tamil Nadu States) and some Australian plants (Adelaide, SA, Australia). Tissue segments from tender shoot or petiole tissues of field-grown plants were generally used. Wherever field plants were not accessible, seedlings/plants raised in glasshouse on soil-based medium were employed. Tissues were used after surface sterilization, which involved two initial rinses in autoclaved distilled water (ADW) containing 0.1% Tween-20 and 5–6 min sodium hypochlorite (2% available chlorine; Fisher Scientific, Mumbai, India) treatment with a change of disinfectant after 3 min, and final six rinses in sterile water. When tender tissues such as coleoptile or hypocotyl of seedlings was used, the duration of chemical treatment (3 min) or the strength of disinfectant was reduced (1% chlorine).