Absolute methanol

Drops of blood were smeared on glass slides. Thick smears were fixed with absolute methanol (Sigma-Aldrich, St. Louis, MO, USA) for one minute, stained with Giemsa (Sigma-Aldrich, St. Louis, MO, USA) in a phosphate buffer (with a pH of 7.2), examined under a light microscope (CH20, Olympus, Tokyo, Japan), and morphologically identified for microfilariae with or without sheath species using the taxonomic key [55 (link),56 (link),57 ].

Blood smears were prepared daily, fixed with absolute methanol (Sigma-Aldrich), and stained with Giemsa (Merck). Parasitemia assessment was performed using the oil 100× objective of a Zeiss Standard 20 microscope (Carl Zeiss LTD, Welwyn Garden City, UK). When the number of parasitized red blood cells reached 0.5%, 200 red blood cells were counted. Lower levels of parasitemia were estimated by considering the parasitized erythrocytes present in 50 fields. The course of infection in each group is displayed as the geometric mean of the parasitemia in each group.

The cell pellet stored in liquid nitrogen was thawed at room temperature and then transferred into a microcentrifuge tube. Distilled water (dH₂O) with pH 10 was added into each tube followed by the addition of absolute methanol (Sigma Aldrich, Saint Louis, MO, USA). The cell suspension was agitated in a tube shaker for 20 min at 11,000× g. Then, the cell suspension was sonicated for one minute at 0 °C. Subsequently, absolute chloroform (Merk Milipore, Burlington, MA, USA) was added into the cell suspension, resuspended, and vortexed. The cell suspension was sonicated again for one minute at 0 °C. The suspension was transferred into a new microcentrifuge tube and centrifuged for 20 min at 11,000× g with 4 °C. After centrifugation, different layers were separated. Polar metabolites were extracted in upper layer and the nonpolar metabolites were extracted in the lower layer. The upper layer and lower layer were collected and transferred to new 2 mL centrifuge tube and mixed thoroughly. The samples were then dried at 0 °C for 120 min using an Eppendorf Concentrator Plus and were vortex-mixed for one minute before they were finally transfer into vials for LC/MS analysis.

The clonogenic assay was performed with V79 cells as described by Louro et al. (2019) [63 (link)]. Briefly, a very low density of V79 cells (50 cells) was plated in each well of a 6-well plate and allowed to attach for approximately 7 h, at 37 °C and 5% CO₂. The cells were then exposed to 1.5, 3, 6.25, 12.5, 25 and 50 µg/cm² nanocellulose (7.2–240 µg/mL) during 17 h at 37 °C, 5% CO₂. For each experiment, negative (non-treated cells) and positive (0.2 µg/mL mitomycin C, Sigma) controls were included. At the end of this time, cells were washed with PBS (Gibco) and were incubated with fresh medium for a further 4 days, at 37 °C and 5% CO₂ to allow colony formation. The wells were then washed with PBS, fixed with absolute methanol (Sigma) and stained with 10% Giemsa (Merck, Darmstadt, Germany). The number of colonies formed was counted and the cloning efficiency (CE) determined using the following equation [63 (link)]: CE = 100 × (no. colonies in negative control/no. of plated cells). The surviving fraction (SF) for each CNF concentration was calculated as follows: SF = (no. colonies formed after exposure/no. of plated cells) × CE/100. The cytotoxicity was determined based on the results from three independent experiments.

Following incubation, the coupons were carefully removed from the growth medium using sterile forceps, gently tapped against the side of the falcon tube to remove excess liquid, and rinsed three times with 25 mL of sterile filtered water to remove loosely-adherent bacteria. Subsequently, the coupons with attached bacterial cells were fixed with 25-mL absolute methanol (analytical grade, >99%, Sigma-Aldrich) for 15 min. Coupons were air-dried for 2 min, and stained with 0.5 % (w/v) crystal violet solution (Sigma-Aldrich) for 15 min, which was followed by three washes with distilled water and air-drying. The dye bound to the biofilm was then dissolved with 25 mL of 33% glacial acetic acid (Sigma-Aldrich) and measured at a wavelength of 590 nm using a spectrophotometer as described previously [21 (link)].

200 cells were seeded in triplicates in 6 well plate. Each 3-4 days, the media was changed until cell colonies were visible by naked eye. Then the media was removed and wells were washed with PBSX1 (Biological industries). Then PBS was aspirated and wells were left to dry. After drying, cells were fixed with absolute methanol (Sigma) for about 15 min at RT. Afterwards, wells were left to dry and then stained using Coomassie blue (Bio-Rad) for about 15 min. Finally, the stain was removed and wells were washed using tap water.