Flat bottom 96 well microplate

HLMVECs harvested from the lung of a single healthy adult donor were purchased from Cell Applications, cultured on surfaces coated with attachment factor solution (AFS, Cell Applications), and maintained in microvascular endothelial cell growth medium (Cell Applications). HUVECs harvested from the umbilical vein of a single healthy neonate were purchased from Cell Applications and maintained in endothelial cell all-in-one growth medium (Cell Applications). All experiments with HLMVECs were performed using passage 4–6 cells. Experiments with HUVECs were performed using passage 3–6 cells. Experimental conditions were maintained in a Heracell VIOS incubator (Thermo Fisher Scientific) using humidified air with 5% CO₂ at 37°C. For the measurement of constitutive Ang-2 release, HLMVECs and HUVECs were grown in flat-bottom, 96-well microplates (Corning) and media collected at the indicated time points. Samples were centrifuged at 10,000g for 10 minutes at 4°C, and harvested supernatant was stored at –80°C. For flow-related experiments, HLMVECs or HUVECs were grown to a confluent monolayer in 0.4 mm height, μ-slide Luer single-channel flow chambers purchased from Ibidi. Endothelial cells were then either maintained in static culture with twice-daily media changes or conditioned under shear stress using a flow system.

ELISA was performed to detect anti-Leishmania antibodies in sera samples [19 (link)]. The flat-bottom 96-well microplates (Corning, USA) were coated with 100 μL/well of purified antigen at a concentration of 5 μg/mL in 0.1 M carbonate/bicarbonate (pH 9.6) buffer by overnight incubation at 4°C. Unbound antigens were removed by washing the plate five times in phosphate buffered saline-Tween 20 (PBST, pH 7.4 containing 0.05% Tween 20). Blocking was performed with 200 μL of 3% nonfat skimmed milk in PBST for 2 hours at room temperature. Then, the wells were washed, 5 times with washing buffer. Diluted serum samples (1 : 100 in PBST) were applied to the plates and incubated for 1 hour at room temperature. The plates were washed as before and 100 μL of 1 : 4000 dilution of horseradish peroxidase-conjugated goat anti-human IgG (Sigma, USA) was added to the plates and incubated for 1.5 hours at room temperature. The plates were then washed as before and incubated with substrate (100 μL/well of 0.4 mg/mL OPD, 0.3% H₂O₂ in 0.1 M citrate buffer, pH 5) for 20 minutes. The reaction was stopped by using 1 N H₂SO₄. The absorbance at 490 nm was checked with a microplate reader (Bio-Tek, ELx800). Positive sera from VL-confirmed cases were applied in each plate. The cutoff point was fixed at 2SD above the mean of control samples.

The E. coli cells were inoculated from glycerol stocks into test tubes containing 3 mL of 10.5 mM glucose-supplemented minimal medium and incubated in a bioshaker (MBR-022UP, Taitec, Japan) at 200 rpm and 37 °C as precultures. The precultures were diluted 1000-fold with fresh minimal medium supplied with 0.105, 0.21, 1.05, 2.1, or 10.5 mM glucose and subsequently loaded into flat-bottom 96-well microplates (Corning, USA) in six wells with locations varied per culture condition, as described previously⁷⁷ . The microplates were incubated in a plate reader (Synergy H1, BioTek, USA) with continuous orbital shaking at 282 cpm and 37 °C. Growth was monitored by measuring the absorbance at 600 nm, and readings were obtained at 30 min intervals for 20–30 h. The growth rate was calculated according to the changes in OD₆₀₀, as described previously⁸¹ .

All of the tested compounds were dissolved in anhydrous
DMSO to
a concentration of 5 mg/mL. Candida strains were
streaked from a glycerol stock onto YPAD agar plates and grown for
24 h at 30 °C. Colonies were suspended in 1 mL of PBS and diluted
to an O.D. of 0.01 at 600 nm with YPAD broth and added into flat-bottom
96-well microplates (Corning) containing a gradient of twofold dilutions
per tested compound with concentrations ranging from 64 to 0.0009
μg/mL. Control wells with no drug and blank wells without yeast
cells containing YPAD only were also prepared. MIC values (Table S5) were determined after 24 h at 30 °C
by measuring the optical density at 600 nm using a plate reader (Tecan
Infinite M200 PRO). MIC values were defined as the point at which
the optical density was reduced by ≥65% compared to the no-drug
wells. Each concentration was tested in triplicate, and results were
confirmed by at least two independent sets of experiments.

Candida strains were
streaked from glycerol stock onto YPAD agar plates
and grown for 24 h at 30 °C. Colonies were suspended in 1 mL
of PBS and diluted to an optical density of 2 × 10^–4 at 600 nm (OD₆₀₀) in flat-bottom 96-well microplates
(Corning) with YPAD broth containing dilutions of each tested compound
at concentrations ranging from 64 to 0.008 μg/mL in twofold
dilutions. Control wells with yeast cells but no drug and blank wells
that contained only YPAD were prepared. MIC values (Table S2) were determined after 24 h at 30 °C by measuring
the OD₆₀₀ using a plate reader (Infinite M200 PRO, Tecan).
MIC values were defined as the point at which the OD₆₀₀ was reduced by ≥75% compared to the no-drug wells. Each concentration
was tested in triplicate, and the results were confirmed by two independent
sets of experiments.

The protein quantification from the C6/36 EVs isolates was performed according to the specification given by the Micro BCA Protein Assay kit (Thermo-Fisher Scientific). The calibration curve standards were performed, in dilutions of 1:2, from a concentrated solution of 2 mg/mL of BSA (Thermo-Fisher Scientific). The blanks, standards, and C6/36 EVs samples (150 µL) were added in triplicate to flat bottom 96-well microplates (Corning) following the addition of 150 µL of the kit work reagent mixture. The plate was covered with sealing tape and incubated at 37 °C for 2 h. The absorbances were measured at 562 nm on the Multiskan Ascent spectrophotometer (Thermo Labsystems) with the Ascent software version 2.6. The average of the 562 nm absorbances of the blank samples was rested from the 562 nm reading of each standard and C6/36 EVs samples. The standard curve was used to determine the protein concentration (mg/mL) of each C6/36 EVs sample.