Infinity 200 pro

CQDs were synthesized by pyrolsis method reported earlier^{67 (link)}. The cytotoxicity of CQDs was assessed in cell culture using MTT assay. Human monocytic THP-1 cells and HeLa cells were seeded at a density of 2 × 10⁴ cells / well in 100 µl RPMI 1640 and DMEM medium in a 96-well plate at 37 °C in CO₂ incubator, containing different concentrations of CQDs (0, 12.5, 25, 50, 100 and 200 μg/mL) for 24 h and 48 h. Thereafter, 20 µL MTT (5 mg/mL) in 200 µl of RPMI-1640 medium was added to each well and incubated for 2 h at 37 °C in CO2 incubator. After discarding the 150 µl media, 100 µl of DMSO was added to dissolve the formazan crystals. Next, the optical density was measured at 540 nm using a microplate reader (TECAN Infinity 200 pro). Three independent experiments were performed, and results were presented as the mean ± SEM. One-way analysis of variance (ANOVA) was done to test the significance of difference in the proliferation of THP1 cells at different doses of CQDs using GraphPad’s Prism5 software.

In order to quantify the proliferation of MC3T3 an AlamarBlue assay was carried out the day after the last stimulation protocol (day 10). Briefly, 300 μL AlamarBlue mixture containing 10%AlamarBlue™ stock solution (ThermoFisherScientific, UK) and 90% cell culture medium was added directly to the samples in a culture plate with electrodes, then incubated at 37 °C for 120 min. Following incubation, three 50 μL replicates of the aliquots were transferred from each sample into a clear 96 well plate. A sample control (media plus AlamarBlue™ reagent) was required on this assay. Fluorescence readings were taken using a TECAN, Infinity 200 Pro microplate reader at 570 nm excitation.

Cells were exposed to the particles as mentioned above. After exposure, cell viability was determined using the CTB cell viability kit (Promega, Madison, USA). For all tests performed, untreated cells were used as a negative and 0.1% Triton X-100 treated cells were used as a positive control. Fluorescence was measured at 590 nm upon excitation at 560 nm using a plate reader (Infinity 200 Pro, Tecan, Groedig, Austria).

For incubation of capsosomes with bacterial cultures, a 96-well plate was prepared with column A containing 95 μL of 5 mg mL⁻¹ SRB-capsosomes and 95 μL TSB, and all other wells containing 95 μL TSB. A serial dilution was performed across the columns with the final column containing no capsosomes. Stationary-phase cultures of S. aureus JE2, S. aureus USA300, and S. aureus USA300 ΔagrA were diluted and inoculated into the 96-well plate to reach a final cell density of ≈1 × 10⁵ CFU mL⁻¹. For incubation of capsosomes with bacterial supernatants, a 96-well plate was prepared containing 50 μL of 5 mg mL⁻¹ SRB-capsosomes and 50 μL buffer in column A, which was serially diluted into 50 μL buffer in all other wells as with the bacterial culture samples. A 50 μL aliquot of bacterial supernatant was then added to each well. For incubation of capsosomes with synthetic PSMs, 200 × 10⁻¹M synthetic PSMs were mixed with 0.05 mg SRB-capsosomes in buffer to a final volume of 100 μL. Samples were measured every 30 min for 17 h using a Tecan Infinity 200 Pro plate reader with an excitation wavelength of 553 nm and an emission wavelength of 585 nm. Triton was then added to column A to form a final concentration of 1 vol%. The samples were incubated for 1 h shaking and then measured again using a plate reader.

Cell proliferation and viability tests were evaluated using AlamarBlue^® reagent (Invitrogen, Carlsbad, CA, USA). According to the manufacturer’s protocol. Subsequently, the absorbance at 570 nm (oxidized) and 600 nm (reduced) (TECAN, Infinity 200 Pro, Männedorf, Switzerland) was recorded.

To determine the antibody titer, an indirect ELISA test was performed as described by El Kojok et al. [36 (link)], with slight modifications. The ELISA plate was coated with 50 μL/well of recombinant antigen (from 1 µg/mL to 0.001 µg/mL) and/or virus sample (from 1:10 to 1:1000) diluted in 0.05 M carbonate buffer at pH 9.6 and incubated overnight at 4 °C. As a negative control, some wells were coated by coating buffer. The wells were washed three times with washing buffer (TBS 0.01 M pH 7.4 containing 0.05% Tween-20; TBST) and after the incubation with 200 μL/well of blocking buffer (TBS supplied with 5% w/v non-fat dried milk), at 37 °C for 2 h, the plate was rinsed three times. After this step, 50 μL/well of polyclonal antibodies, anti-ASFV (1 μg/mL) diluted in TBS, 1% non-fat dried milk, and 0.05% v/v TWEEN 20 buffer, were incubated at 37 °C for 2 h. The plate was rinsed (three times), 50 μL/well of goat anti-rabbit IgG-HRP antibody (0.5 μg/mL), was added, and the wells were incubated for 1 h at 37 °C. Finally, the enzyme substrate solution (TMB) was added (100 μL/well), and the wells were incubated at 37 °C, then the color development was quenched by adding stopping solution HCl (2.5 M 50 μL/well) after 10 min. A Tecan Infinity 200 Pro (Tecan, Männedorf, Switzerland) micro-plate reader was used to measure the absorbance at 450 nm.

A fluorescence-based kit for determination of GSH and GSH/GSSG ratio was used according to manufacturer's instructions (BioVision). Briefly, cells were spun, washed with PBS and lysed in cell lysis buffer. 1 μg of total protein was loaded into 25 microliters of assay buffer in a 384-well plate. 25 microliters of the glutathione assay mixture (GAM) or total glutathione assay mixture (TGAM) mixture was added to samples, and reduced or oxidized glutathione standard curves plotted. Fluorescence was measured at the excitation wavelength of 480 nm and emission wavelength at 520 nm using Tecan infinity 200 PRO.