Incu mixer mp

MIC was determined by microbroth dilution method as described. Briefly, bacterial cells (0.5 McFarland turbidity) were mixed with nanomaterials (ZO, 2NZO, and 5NZO) varying from 10 to 250 μg mL⁻¹ range in a 96-well microplate. The resulting mixtures were incubated at 37 °C for 16 h with 500 rpm shaking using Incu-Mixer™ MP (Benchmark Scientific). The nanomaterial concentration that completely inhibit the bacterial growth was defined as MIC value.

SS
blood samples were diluted 1:1000 with a modified HEMOX solution,
pH 7.4, containing N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic
acid (30 mM), sodium chloride (135 mM), potassium chloride (5 mM),
calcium chloride (0.9 mM), magnesium chloride (0.5 mM), iron(II) chloride
(9 μM), and glucose (5 mM) in water. Diluted SS blood (20 μL/well)
was aliquoted into 384-well plates (CellCarrier 384-well Ultra microplates,
PerkinElmer, Waltham, MA).
Assay plates, a plate shaker (Incu-Mixer
MP, Benchmark Scientific, Sayreville, NJ), and an oxygen meter (MiniOx
250E, Ohio Medical, Gurnee, IL) were placed in a chamber made of an
inflatable polyethylene bag (AtmosBag, MilliporeSigma, St. Louis,
MO, Figure S2). The chamber was purged
with a mixture of nitrogen (purity 99.9%) and air to maintain a concentration
of oxygen gas of 3.9–4.1%, and the plates were shaken (1000
rpm) within the chamber at 37 °C for 1 h. After incubation, samples
were fixed with 20 μL/well of 2% glutaraldehyde in phosphate
buffer (Electron Microscopy Sciences, Hatfield, PA), sealed, and shaken
in the chamber at 37 °C for 15 min. The plates were subsequently
removed from the chamber and centrifuged at 1000 rpm for 1 min to
sediment RBCs to the bottom of the plates for the subsequent assessment
of RBC appearance.

The production of ROS by E. coli ATCC 25922 after treatment with ZnO nanorods was evaluated based on the previous reports.^{33 (link)} Briefly, the adjusted bacterial culture (0.5 McFarland turbidity) in PBS buffer by Sensititre™ Nephelometer (Thermo Scientific) was treated with different concentrations of nanomaterials corresponding to 0, 125, 200, and 250 μg mL⁻¹ in the presence of 2′,7′-dichlorodihydrofluorescin diacetate (DCFH-DA) (Sigma-Aldrich) at a final concentration of 30 μM in PBS buffer. The mixtures in 96-well plate were incubated at 37 °C with vigorous shaking (500 rpm) using Incu-Mixer™ MP (Benchmark Scientific) for 45 min. Then, the fluorescence intensity was measured by a FLUOstar Omega (BMG Labtech) with an excitation and emission wavelengths of 485 and 520 nm, respectively. Untreated bacterial culture was used as a negative control and the background fluorescence of PBS and autofluorescence of the bacterial cells incubated without the probe was measured to calculate the net fluorescence emitted from the assay itself. The data was further analyzed using MARS Data Analysis software (ver. 3.02 R2, BMG Labtech) and the relative ROS production of samples treated with increasing concentrations of NPs was compared to non-NP treated samples from triplicate experiments with p < 0.05.