Ae30 31

Smear preparation started by opening the received vials. A small piece (20–50 mg) of the brain was picked up with a tip and placed on a wooden spatula. An imprint of the sample was made by pressing a glass slide on the piece of the brain over the spatula. The smear was fixed in high-grade cold acetone. 0.1 ml of clarified anti-rabies nucleocapsid conjugate (Bio-Rad, France) was placed on the sample, and the sample was incubated at 37°C for 30 minutes. Following incubation, the smear was rinsed twice with distilled water after several minutes of being stored in PBS. The stained samples were then analyzed under a fluorescent microscope (Motic AE30-31, Spain) for the presence of Negri bodies [15 (link)–17 (link)].

Cytotoxic activity was measured as decreased viability on treated HL-60 cells. For testing, treated and non-treated cells were counted every 24 h over 3 days in order to determine the cell growth curve. Cell viability was assessed by the Trypan Blue dye (Sigma-Aldrich, St. Louis, MO, USA, T8154) exclusion test using a hemocytometer under a light inverted microscope (AE30/31, Motic).
HL-60 graphs illustrate the sample concentration-cell response relationship with respect to the cell viability and DNA damage and distribution. Cytotoxic effect evaluation was determined during the culture treatment period, establishing a growth curve and estimating the half maximal inhibitory concentration (IC₅₀) for each treatment by regression. Viability curves of leukaemia cells are presented as a survival percentage normalized to a percentage of the control at 72 h growth (control maximum exponential phase) and plotted as viability mean ± standard error of at least three independent experiments for each treatment and concentration.

HL-60 cells were placed in 96-well culture plates (2 × 10⁴ cells/mL) and treated for 72 h with the lyophilized white and black garlic at different concentrations (4 mg/mL, 2 mg/mL, 1 mg/mL, 0.5 mg/mL, 0.25 mg/mL, 0.12 mg/mL, 0.06 mg/mL, 0.03 mg/mL, and 0.015 mg/mL for white garlic and 4 mg/mL, 2 mg/mL, 1 mg/mL, 0.5 mg/mL, and 0.25 mg/mL for black garlic samples). This wide range of concentrations was intended to estimate the inhibitory concentration 50 (IC₅₀).
Cell viability was determined by the trypan blue dye (Sigma, T8154) exclusion test. Trypan blue solution was added to the cell cultures at a 1:1 volume ratio and 20 μL of cell suspension were immediately loaded into a Neubauer chamber. Cells were counted with an inverted microscope at 100× magnification (AE30/31, Motic, Wetzlar, Germany). Curves were plotted as survival percentages with respect to the control growing at 72 h. At least three independent repetitions were carried out.