96 well flat bottomed tissue culture plate

The cell viability was assessed using the 2-(2-m- ethoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4disulfopheny)-2H-tetrazolium, monosodium salt cell counting kit-8 (CCK-8) assay (Bestbio Biotechnology, Shanghai, China). Fresh cells were seeded in 96-well flat-bottomed tissue culture plates (Corning Inc., Corning, NY, USA) at a concentration of 4×10³ cells/well with complete culture medium and incubated for 24 h. Following two washes with phosphate-buffered saline (PBS), the cells were incubated in 100 μl culture medium containing 3, 6, 15, 30 or 60 μM TMPyP₄ for 4 h. Next, cells at each concentration were exposed to the single laser at an energy density of 0, 3, 6 and 12 J/cm², respectively. Following irradiation, the cells were incubated in fresh medium for an additional 24 h at 37°C prior to the CCK-8 assay. A total of 10 μl CCK-8 and 100 μl RPMI-1640 culture medium was then added to each well, and following incubation for 1 h at 37°C, the optical densities of the samples were measured directly using a spectrophotometric microplate reader (Beyotime Institute of Biotechnology, Haimen, China) at a wavelength of 450 nm. Each experiment was performed in triplicate and repeated five times.

The cytotoxic effect of antibacterial extracts was assessed using the MTT assay as described by Mosmann, [27 (link)], targeting normal monkey kidney Vero cells ATCC CRL1586 cultured in complete medium containing 13.5 g/L DMEM (Gibco, Waltham, MA USA), 10% fetal bovine serum (Gibco, Waltham, MA, USA), 0.21% sodium bicarbonate (Sigma–Aldrich, New Delhi, India), and 50 μg/mL gentamicin (Gibco, Waltham, MA, USA). Essentially, Vero cells at 5 × 10³ cells/200 μL/well were seeded into 96-well flat-bottomed tissue culture plates (Corning, USA) in complete medium. Fifty microliters of serially diluted extract solutions (≤100 μg/mL) were added after 24 h of seeding, and the cells plus test substance were incubated for 48 h in a humidified atmosphere at 37°C and 5% CO₂. DMSO (0.4% v/v) was added as a negative control (100% growth). Twenty microliters of a stock solution of MTT (5 mg/mL in 1x phosphate-buffered saline) was added to each well, gently mixed, and incubated for an additional 4 h. After spinning the plate at 1500 rpm for 5 min, the supernatant was carefully removed, and 100 μL of 100% DMSO (v/v) was added to dissolve the formazan. The plate was read on a microtiter plate reader Infinite M200 (TECAN, Männedorf, Switzerland) at 570 nm. The 50% cytotoxic concentrations (CC₅₀) of extracts were determined by analyzing the dose–response curves.

The cytotoxic effect of antiplasmodial extracts and fractions was assessed using the MTT assay [10 (link)], targeting HEK239T cells cultured in complete medium containing 13.5 g/L DMEM (Gibco, Waltham, MA USA), 10% fetal bovine serum (Gibco, Waltham, MA USA), 0.21% sodium bicarbonate (Sigma-Aldrich, New Delhi, India) and 50 μg/mL gentamicin (Gibco, Waltham, MA USA). Essentially, HEK239T cells at 5 × 10³ cells/200 μL/well were seeded into 96-well, flat-bottomed, tissue culture plates (Corning, USA) in complete medium. Fifty µL of serially diluted extracts and fractions solutions (≤ 200 µg/mL) were added after 24 h of seeding and then incubated for 48 h in a humidified atmosphere at 37 °C and 5% CO₂. DMSO was added as positive inhibitor at 10% v/v. Twenty µL of a stock solution of MTT (5 mg/mL in 1× phosphate-buffered saline) were added to each well, gently mixed and incubated for an additional 4 h. After spinning the plate at 1500 rpm for 5 min, the supernatant was carefully removed and 100 μL of DMSO (stop agent) was added. Formazan formation was read on a microtiter plate reader (Versa Max Microplate Reader, Molecular Devices, USA) at 570 nm. The 50% cytotoxic concentrations (CC₅₀) of extracts and fractions were determined by analysis of dose–response curves. Selectivity indices (CC₅₀/IC₅₀) were calculated for each extract and fraction.

A lymphocyte proliferation assay (LPA) was carried out to evaluate cellular immunity against PvGAMA. First, PBMCs were isolated from patients who were acutely infected with P. vivax and those who had been in recovery for 8–10 weeks using Ficoll-Hypaque (Stemcell Technologies, Vancouver, Canada). PBMCs were then washed twice with incomplete Roswell Park Memorial Institute (RPMI) 1640 medium. PBMCs were used for the LPA when viability was greater than 90%. PBMCs were suspended in complete RPMI 1640 medium containing 10% fetal bovine serum (FBS), and 2.5  ×  10⁵ PBMCs were added into each well of a 96-well flat-bottomed tissue culture plate (Corning Inc., New York, NY, USA). The cells were then stimulated by 10 μg/mL of purified rPvGAMA or rPvDBPII. The complete RPMI 1640 medium and PBMCs stimulated with 2% v/v of phytohaemagglutinin (PHA) were used as negative and positive controls, respectively. Cells were cultured for 96 h at 37 °C under 5% CO₂. After 96 h, the culture supernatant was harvested for cytokine detection.

PR8 virus TCID₅₀ infectivity value and lung tissue viral burden were determined as endpoint serial dilutions of virus or tissue homogenates at which 50% of the cells were infected using Madin-Darby canine kidney (MDCK) cells. Lungs obtained from animals 2, 3, or 4 days after INFV A exposure were homogenized using an Omni TH Homogenizer (Omni International, Kennasaw, GA, USA) and Omni tissue disposable generators in Lebovitz L-15 medium (Gibco Life Technologies, Grand Island, NY, USA) containing antibiotic-antimycotic mixture (Invitrogen, Grand Island, NY, USA). After clarification by centrifugation, supernatant was added (20 µL/well) to 4 replicate wells of a 96-well flat-bottomed tissue culture plate (Corning, Tewksbury, MA, USA) containing MDCK cells in logarithmic (log) phase growth, and serially diluted 10-fold for a total of 8 dilutions. The plates were incubated at 37 °C in 5% CO₂ for 4 days and cells inspected microscopically. Cytopathic effect was noted and confirmed visually by adherence of turkey RBC. The results of viral load assessment were expressed as TCID₅₀ per gram of tissue [24 (link)].
To quantify lung viral load, four mice per group were euthanized on day 3 following PR8 H1N1 virus challenge and on days 2 and 4 following pH1N1 or H5N1 virus challenge.