Multiskan fc

To measure total IgG levels in plasma, a 96-half-well NUNC Maxisorp™ plate (Nalgene) was entirely coated overnight at 4°C with 25 μL of goat anti-human F(ab)′2 (1:1000). As above, plates were washed in PBS-T and blocked for 1 hat RT in assay buffer. 25 μL of serial dilutions of patient plasma (1:100 to 1:10⁷) were added in duplicates to the plate alongside known concentrations of IgG in triplicates. As above, after 2 h of incubation at RT, plates were washed with PBS-T and 25 μL AP-conjugated goat anti-human IgG was added and then incubated for 1 hat RT. Plates were washed with PBS-T, and 25 μL of AP substrate added. ODs were measured using a Multiskan™ FC plate reader at 405 nm and total IgG titers interpolated from the IgG standard curve using 4 PL regression curve-fitting on GraphPad Prism 9.

Biofilm formation assays were performed in 96-well polystyrene microtiter plates, as previously described (12 (link)) with minor modifications. The single colony on the blood plate was shaken overnight in 3 mL of fresh LB broth medium at 37°C. Then, the culture was adjusted to 0.5 McFarland with sterile normal saline and further diluted by 1:100 in LB broth and dispensed in a 96-well microtiter plate with 32, 64, or 128 μg/mL resveratrol and 1 μg/mL colistin either alone or in combinations. The 96-well plates were incubated at 37°C for 24 h. Then the cell suspension was removed, and the plates were washed twice with 1× phosphate-buffered saline (PBS) (Sigma–Aldrich, Milan, Italy) and inverted to dry at the room temperature. Next, 200 μL of 1% crystal violet (CV) solution (Beijing Solarbio Biotechnology Co., Ltd., China) was added to the wells for 15 min. After staining, CV was removed, and the wells were washed thrice with 1× PBS. The plate was dried naturally at room temperature, and the bound CV was solubilized by adding 200 μL of ethanol–acetone (96:5 vol/vol) solution. The absorbance of CV was read at 595 nm on a microplate reader (Multiskan FC). All tests were performed in triplicate.

The Caspase-3 activity detection kit (Cat.No. C1116, Beyotime, Shanghai, China) was adopted to test the caspase-3 activity in chondrocytes. 40 µL detection buffer, 50 µL sample, and lysis buffer were successively added to the 96-well plates, followed by the addition of 10 µL Ac DEVD pNA (2 mM) for treatment. After incubation at 37 °C for 60–120 min, the absorbance of Caspase-3-catalyzed pNA in different samples was monitored at 450 nm with a Thermo microplate reader (Multiskan FC).

The anticoagulant activity of the polycarboxylates and heparin was evaluated using Test Team Heparin S (Sekisui Medical, Tokyo, Japan) according to the manufacturer’s instructions. Each polymer solution (32 μL) was combined with plasma (4 μL) and anti-thrombin III solution (4 μL) in 96-well plates (Thermo Fisher Scientific) and incubated for 5 min at 37 °C. Factor Xa solution (20 μL) was then added to each well and incubated for 30 s at 37 °C. A chromogenic substrate solution (40 μL; substrate: S-2222) was added to each well and incubated for 3 min at 37 °C. Finally, the absorbance at 405 nm was measured on a Multiskan FC plate reader.

The cytotoxicities of pentamidine, linezolid, their combination, and 2.5% (vol/vol) DMSO, were tested on RAW264.7 cells (Procell Life Science and Technology Co., Ltd., China) via the Cell Counting Kit-8 (CCK-8) method. 2 × 10⁵ cell lines were seeded at 100 µL/well into 96-well tissue culture plates (Corning, USA) and incubated for 12 h. Following treatment with different concentrations of pentamidine and/or linezolid as well as 2.5% (vol/vol) DMSO (the control group with cells only), the cell lines were incubated in the presence of CO₂ at 37°C for 24 h. After that, 10 µL of CCK-8 reagent (MedChemExpress, USA) were added and incubated for 2 h at 37°C. The absorbance was measured at 450 nm using a microplate reader (Multiskan FC).

CCK-8 test was performed for the detection of cell proliferation in hydrogels. Specifically, the cell/hydrogel constructs were submerged in 500 µl of fresh serum-free medium with 10% CCK-8, then placed at 37°C and cultured for 4 h. The absorbance of the reacted solution at 450 nm was measured by a Microplate Reader (Multiskan FC, USA). Three parallel samples in each group were detected, and the results were averaged.

The Supo HHS-6 electro-thermostatic water bath and the Evela N-1200A rotary evaporator were used. The freeze-drying method was operated by Biosafer Biosafer-10A and Christ Alpha 1–2 LD plus vacuum freeze-dryers. The size distribution and zeta potential were determined using a Malvern Zetasizer nano ZS analyzer. The UV used a Shimadzu UV-2600 UV-vis spectrophotometer, and the ATR-FTIR used a Perkin Elmer Spectrum two infrared analyzer. The JEOL JEM-2010HR was operated for TEM observation.
The cell cycles were performed by a Beckman CytoFLEX S flow cytometry analyzer. Cell proliferation was determined with a Multiskan FC ThermoFisher microplate reader. The quantitative RT-PCR was conducted with 7500 apparatus, Applied Biosystems (Waltham, MA, USA).