Microplate reader

Quartz Crystal Microbalance with Dissipation Monitoring (QCM-D, E4) (Biolin Scientific AB/Q-Sense, Gothenburg, Sweden) was used for affinity analysis; Real-time quantitative PCR system (TL980) (Xi’an Tianlong Science and Technology Ltd., Xi,an, China) was applied for monitoring the richness of library; PCR (T960) (Heal Force Bio-meditech Holdings Limited, Shanghai, China) was used for amplifying; F-7000 fluorescence spectrophotometer (Hitachi, Tokyo, Japan) was used for fluorescence detection; Microplate reader (Flash spectrum, Shanghai, China) was used for recording the absorbance; Micronucleic acid quantifier 3000 (Hangzhou Allsheng Instruments Co, Ltd., Hangzhou, China) was applied for ssDNA quantitation.

The cytotoxicity assay was conducted using the CyQUANT™ MTT Cell Proliferation Assay Kit from Invitrogen (Cat. No. V13154). Neutrophils were seeded at a concentration of 2 × 10⁵ cells per milliliter in conical tubes. The control and antioxidant-supplemented neutrophils (GSH + NAC and ALL) were pre-incubated for 60 min with their respective media (see materials and methods 4.2). After the pre-incubation period, the neutrophils were washed twice with PBS 1× to remove the media. Then, 100 µL of freshly prepared RPMI 1640 media alone was added to each well of a Nunc Flat-bottom microplate from Thermo Fisher (Cat. No. 168055), with a concentration of 2 × 10⁵ cells per well. Subsequently, 10 µL of the CyQUANT™ MTT reagent was added to each well. The negative control of RPMI 1640 + MTT was included, as indicated by the manufacturer’s protocol. The plate was incubated for 3 h at 37 °C. After the incubation period, the plate was centrifuged for 10 min at 4 °C and 2000 rpm. Then, 85 µL of the total volume was carefully removed, and 50 µL of dimethyl sulfoxide (DMSO) was added to each well. The plate was incubated for 15 min, and each well was resuspended prior to reading. Absorbance readings were performed using a microplate reader (ALLSHENG) at 540 nm.

To evaluate the antioxidant effect in both medium and neutrophils, we utilized the Total Antioxidant Capacity Assay Kit from Abcam (Cat. No. ab65329, Cambridge, UK) following the manufacturer’s protocol without the protein mask. The TAC was assessed in the culture media components: RPMI 1640 alone, AS alone, and RPMI 1640 + AS, following the manufacturer’s instructions for liquid samples. For the evaluation of TAC in neutrophils, we seeded 1 × 10⁶ neutrophils and followed the same protocol described in the section of NETosis assay for the controls and the combination of antioxidants GSH + NAC and ALL. After inducing NETosis, in all cases, the cell suspensions were washed twice with cold PBS 1× to remove the media. Subsequently, the samples were resuspended in 100 µL of ddH₂O containing 0.05% Triton X-100 (Cat. No. 85111, Thermo Fisher). The resulting homogenate was then incubated on ice for 10 min. After incubation, the samples were centrifuged at top speed for 5 min at 4 °C. The supernatant was collected, and the assessment was carried out according to the instructions provided in the kit. The output of the TAC assay was measured using a microplate reader (ALLSHENG, Hangzhou, China) at 570 nm.

To evaluate nitrite + nitrate production in neutrophils, we used the Nitrate/nitrite Colorimetric Assay Kit from Cayman (Cat. No. 780001, Ann Arbor, USA). Neutrophils were seeded at a concentration of 1 × 10⁶ cells per milliliter in 1.5 mL conical tubes. The samples were pre-incubated for 60 min with their respective media as described in the media preparation section for controls and the antioxidant combinations (GSH + NAC and ALL). After pre-incubation, the cell suspensions were washed twice with cold PBS 1×, and the protocol described for NETosis assay was conducted. Subsequently, the cell suspensions were sonicated with 5 cycles of 5 s with intervals of 5 s off. The cells were centrifugated at 14,000 rpm for 30 min, and the supernatant was collected. The nitrite/nitrate assay was conducted using a volume of 80 µL of the processed samples, following the manufacturer’s protocol provided with the kit. The absorbance was measured at 550 nm using a microplate reader (ALLSHENG).

The qualitative antibacterial activity of electrospun webs were evaluated according to standard AATCC 147-1998 through a disc diffusion assay against E. coli and S. epidermidis. First, 1 mL of overnight-precultured bacterial inoculums in nutrient broth was transferred to a new 10 mL fresh broth and incubated for 5 h at 200 rpm at 37 °C. The cultures were then diluted to an OD₆₀₀ of 0.7 corresponding to 3.5 × 10⁹ and 1.8 × 10⁹ CFU mL⁻¹ for E. coli and S. epidermidis, respectively, via a microplate reader (Allsheng, Hangzhou, China). The diluted culture was then spread over the entire surface of the nutrient agar plate using a spreader. Electrospun webs were cut into 20 mm diameter discs and then placed on the inoculated nutritional agar plate’s surface. The antimicrobial activity of the electrospun webs were recorded in terms of inhibition zone diameter (mm) using a manual caliper after plates were incubated for 24 h at 37 °C. Experiments were replicated for each sample. For the morphology of bacteria, samples were observed by SEM. Bacteria cells were fixed by osmium tetroxide vapor (2% w/v) for 24 h.

1 × 10⁴ cells/well CAL27 and SCC25 cells were inoculated onto the 96-well plates for 12 h. Next, 10 μL of MTT (0.5 mg/mL, Beyotime, Shanghai, China) was supplemented into per well. 4 h later, medium was replaced by 100 μL of dimethyl sulfoxide (Thermo Fisher Scientific, Waltham, MA, USA). The absorbance was assessed using a microplate reader (Allsheng, Hangzhou, China) at 570 nm.