Trypan blue

Ham's F12, DMEM (high glucose) and RPMI 1640 medium, 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), 2′,7′-dichlorofluorescin diacetate (DCFDA), Neutral red dye, dimethyl sulfoxide (DMSO) were obtained from Sigma Chemicals, Bangalore, India. Fetal Bovine Serum (FBS) was purchased from Invitrogen, USA. Glacial acetic acid, absolute ethanol, ethylene diamine tetra acetic acid (EDTA) was procured from Merck, India. Trypan blue was purchased from HiMedia Labs, India. ELISA kits for human TNF-α, IFN-γ, IL-6, IL-8 and IL-5 were purchased from Krishgen Biosystems, Mumbai, India. ELISA kits for human Involucrin and Filaggrin were purchased from Cloud-Clone Corp., USA. All the molecular biology reagents for PCR were obtained from Bio Rad, USA. Virgin Coconut Oil was obtained from Central Plantation Crops Research Institute (CPCRI), Indian Council of Agricultural Research, Government of India, Kasaragod, India.

For bright field microscopic imaging, cells were cultured at density of 2 × 10⁴ cells/plate in 6 cm culture dishes, treated with flavonoids enriched fractions from both the plants and then images were captured using Olympus (CKX41) microscope at 20 X magnification. To assess the number of dead cells versus the number of live cells, dye exclusion assay using Trypan Blue was performed. This assay is built on the principle that living cells possess intact cell membranes that exclude certain dyes such as Trypan Blue, whereas dead cells lose this capacity and hence retain the dye within the cell [8 ]. 0.4% of the concentration of Trypan Blue (procured from HiMedia) was prepared using molecular grade water. Equal volumes of cell suspension (diluted) and 0.4% of Trypan Blue solution were mixed in a tube and incubated at room temperature for 5–10 min. 20 μl of this mixture was loaded onto the Biorad Chamber slide and the number of live and dead cells were analyzed using Biorad TC20 Automated cell counter.

The L-15 Medium (Leibovitz's), Medium 199, CollagenaseType I, Gelatin solution (2%), Antibiotic-Antimycotic solution (100X), HiEndo ^XL Endothelial Cell Expansion medium (Reduced serum), Heparin sodium salt, Trypan Blue, Trypsin solution (0.25%) were procured from Himedia. Fetal Bovine Serum (FBS), Poly-l-Lysine, Thrombin from Bovine Plasma and 2, 3 bis (2-methoxy- 4 nitro 5- sulfophenyl) 2H- tetrazolium- 5 carboxanilide inner salt (XTT) were purchased from Sigma (St. Louis, MO,USA). Other chemicals are of analytic grade. Cell culture plastic wares were purchased from Axiva Sichem Pvt. Ltd. (New Delhi, India).

The method developed by Phillip and Hayman (1970) was used to evaluate rice root colonization by AMF (Ganeshamurthy et al., 2017 ). To commence the procedure, freshly collected root samples were delicately washed to remove soil attached to the root surfaces. The samples were then submerged in a 10 % KOH solution and autoclaved for 15 min at 121 °C. Following autoclaving, the KOH solution was decanted, and the treated roots were rinsed with tap water three times until no brown color appeared in the rinsed water. The root samples were then immersed in a 2 % HCl solution for 5 min, without rinsing with water. The HCl solution was decanted, and the root samples were stained with 0.05 % trypan blue (HiMedia, India) in lacto-glycerol [lactic acid (400 mL) + glycerol (400 mL)+ water (100 mL)]. The stained samples were then autoclaved for 15 min at 121 °C, after which the staining solution was decanted, and the roots were de-stained with lacto-glycerol solution to remove excess stain. The resulting segments were observed under a compound microscope (Radical RxLr-4, India), and the method proposed by McGonigle et al. (1990) (link) was used to calculate the percentage of root colonization.
AMF root colonization was calculated using the formula: