Rpmi 1640 medium

HUVEC were purchased from HiMedia Laboratories (Mumbai, India) and maintained in tissue culture flasks coated with 0.5% gelatin in HiEndoXL Endothelial Growth medium (HiMedia Laboratories, Mumbai, India) supplemented with HiEndoXL Endothelial Growth Supplement (HiMedia Laboratories, Mumbai, India) and 1× antibiotic antimycotic solution (HiMedia Laboratories, Mumbai, India). The cultures were maintained at 37 °C and 5% CO₂. Logarithmically growing cells (passages 2–6) were used for all experiments. Human monocyte (THP-1) cell line was obtained from National Centre for Cell Science (NCCS), Pune, Maharashtra, India and maintained in RPMI-1640 medium (HiMedia Laboratories, Mumbai, India) supplemented with L-glutamine (2 mmol/l), 10% FBS (Gibco, Thermo Fisher Scientific, USA) and 1× antibiotic–antimycotic solution (HiMedia Laboratories, Mumbai, India) in a humidified atmosphere with 5% CO₂ at 37 °C. For the induction of cell differentiation, THP-1 cells (1.5 × 10⁶ per ml) were seeded in serum-free RPMI-1640 with 50 nM PMA for 24 h. After incubation, non-adherent cells were removed by aspiration, and the adherent THP-1 derived macrophages (TDMs) were washed with PBS before experimental treatments.

Human embryonic kidney cells (HEK293T) and human lung cancer cells (A549) were purchased from NCCS, Pune and cultured in Dulbecco’s Modified Eagle’s Medium (Himedia). Human splenic marginal zone lymphoma SSK-41 cell line was established and cultured in RPMI 1640 medium (Himedia) and B-cell lymphoma cells (JM1) was purchased from NCCS, Pune and cultured in Iscove’s Modified Dulbecco’s Medium (Himedia) supplemented with 10% fetal bovine serum (Gibco, Thermo fisher Scientific, Waltham, MA, USA) and 1% penicillin/streptomycin solution in a humidified incubator at 37°C and a 5% of CO₂ atmosphere [41 (link)].

This research was conducted using C. albicans ATCC 10231 (Microbiologics, United States), a reference strain commonly used for evaluating antifungal and antibiofilm agents. Experiments were conducted with two clinical strains of C. albicans [CA i16 (GenBank No. MG757722.1) and CA i21 (GenBank No. MG757724.1)] which were isolated from sputum samples of patients. The clinical strains were obtained from University of Madras, India. These cultures were routinely maintained on potato dextrose agar (PDA) (HiMedia, India). For liquid cultures, a single colony was picked from PDA, transferred to potato dextrose broth (PDB) and incubated for 24 h at 30°C and 120 RPM in a temperature controlled orbital shaker. Cells harvested from PDB were used for growth and biofilm experiments.
Filter sterilized RPMI 1640 medium (L-Glutamine, phenol red, 2 g l^–1 glucose and 0.165 mol l^–1 MOPS buffer, pH 7.0) (Part No. AT180, HiMedia, India) was used for biofilm experiments. Cultures were grown in PDB for 24 h, pelleted by centrifugation, re-suspended in RPMI 1640 and adjusted to desired cell density for performing biofilm experiments. For determining the mechanism of action, cells were re-suspended in phosphate buffered saline (PBS).

A human-monocyte-like cell line (U937) was purchased from National Centre for Cell Science (NCCS), Pune, India. In addition, peripheral blood mononuclear cells (PBMCs) were freshly isolated using the blood of healthy donors. The PBMCs’ isolation process is described later in this section. Both cell types were maintained in Roswell Park Memorial Institute (RPMI) 1640 medium (HiMedia Laboratories, LLC, Kennett Square, PA, USA) + 10% Fetal Bovine Serum (FBS) (HiMedia Laboratories, LLC, Kennett Square, PA, USA) + 2 mmol/L L-glutamine + 1% penicillin + streptomycin (Sigma-Aldrich, St. Louis, MO, USA) at 95% relative humidity and 5% CO₂ at 37 °C.

Chicken splenocytes were isolated from spleens collected from healthy birds, under laminar air flow as per standard procedure [13 (link)]. Lymphocytes were separated by density gradient centrifugation (Hisep-LSM, HiMedia, India) as per the method described by Rose and Friedman [14 ]. Percentage cell viability was determined by 0.1% Trypan blue dye exclusion test using hemocytometer [15 (link)], and final cell count was adjusted to 10⁷ cells/ml in RPMI-1640 medium with antibiotic and antimycotic solution supplemented with 10% Fetal Bovine Serum (all HiMedia, India).

LC-MS grade water, methanol, acetonitrile, formic acid, Zorbex UPLC C₁₈ Silica Column, Agilent 6520 QTOF instrument, acetylcholinesterase from electric eel, butyrylcholinesterase from equine serum, amyloid beta protein (Link biotech), thioflavin T, Congo red dye, reserpine, and ajmalicine were purchased from Sigma Aldrich, India. MAO-B inhibition kit by Biovision. BACE-1 activity assay kit by Sigma Aldrich. RPMI 1640 medium, FBS, PBS, and other tissue culture grade chemicals were purchased from Himedia, India. PC12 (rat pheochromocytoma) cell line was obtained from NCCS, Pune, India. All other reagents used were of analytical grade. All preparations used in tissue culture experiments were filtered through 0.45 µm Axiva 25 mm CA filter. Ethical approval was obtained from GGS Indrapratha University for all the cell culture work done in proper confined facilities.