Model elx800

The in vitro cytotoxicity of our nanomicelles was assessed by the standard MTT assay. Typically, HCT-116 and HCT-8 cells were seeded in a 96-well plate with a seeding density of 1 × 10⁵ cells per well for 12 hours. Then the cells were incubated with free UA or FUP with various concentrations. After 24 hours of incubation, the medium was removed, and 100 μL of MTT (0.5 mg mL⁻¹ in PBS) was added for further incubation for 4 hours, the purple-blue formazan precipitate was dissolved in 100 μL of DMSO. The absorbance of the solution in each well was measured using an ELISA reader (Model ELX800, BioTek, USA) with the wavelength at 490 nm. The proliferation of cells was determined by the absorption intensity. Cell viability was calculated as follows: Cell viability (%) = OD_test/OD_control × 100%, where OD_test and OD_control are the absorbance values for the treated cells and the untreated control cells, respectively. The OD_test and OD_control values were obtained after subtracting the absorbance of DMSO. All tests were performed in quadruplicate. Cell viability graphs were plotted as the UA concentration. Meanwhile, the in vitro cytotoxicity of blank vehicles of F127/C₁₈-PEI was also evaluated on NIH3T3, HCT-116 and HCT-8 cells by using the same methods.

Tissue homogenates of the ipsilateral hippocampus and sensorimotor cortex were separated by centrifugation for 20 min at 3000 r.p.m. Inflammatory cytokines IL-1β and IL-10 levels were detected by ELISA kits according to the manufacturer’s protocols (Xitang, Shanghai, China). In brief, samples were coated with 100 μl capture antibody at 4 °C. Followed three washes, using 200 μl assay diluents to block at room temperature for 1 h, then 100 μl diluted IL-1β or IL-10 standards and test samples were added and incubated for 1 h at 37 °C. Following repeated washes, each well was added with the substrate and incubated for 20 min at room temperature. The quantitative absorbance was determined at 450 nm using an ELISA reader (BioTek, Model ELX800, USA) according to IL-1β or IL-10 protein standards.

Cell proliferation was detected by cell counting kit-8 (CCK-8) assay, 5-ethynyl-2′-deoxyuridine (EdU) incorporation test, and nucleoprotein Ki-67 and proliferating cell nuclear antigen (PCNA) expression analysis. First, for the CCK-8 experiment, cells were seeded into a 96-well plate with 10% FBS DMEM for 24 h. Then, the medium was changed with serum-free DMEM for CCK-8 kit (Beyotime Biotechnology, Shanghai, China) detection. A microplate reader (Model ELX800, BioTek, Vermont, USA) was used to determine the absorbance at 450 nm, which reflects cell viability and proliferation. Second, cells were seeded into a 24-well plate for EdU assessment. After 48 h of transfection, DNA synthesis was examined with an EdU incorporation assay (Guangzhou RiboBio, Guangzhou, China). The proportion of EdU-positive cells was analysed by fluorescence microscopy (DP70, Olympus Optical, Tokyo, Japan). Finally, cell proliferation was evaluated by Ki-67 and PCNA expression. Ki-67 is a nucleoprotein that is a marker of tumour proliferation [28 (link)]. PCNA acts on chromatin and participates in all aspects of the DNA replication chain [25 (link)]. The expression levels of Ki-67 and PCNA can be used to evaluate the status of cell proliferation.

Biofilm formation of rugose and smooth morphotypes was measured by crystal violet assay (O’Toole, 2011 (link)). An overnight culture of smooth and rugose morphotype was grown in TSB and was diluted to achieve a final inoculum level of ∼10⁶ CFU/ml. Biofilm was developed after inoculation of 200 μl TSB culture containing 6 log CFU/ml of bacteria in individual wells of a 96 well polystyrene microtiter plate (SKU 229190, Celltreat Scientific Product, Pepperell, MA) and incubation at 25°C for 48 h. After 48 h of incubation, bacterial inoculum was aspirated out from each well of a plate. Thereafter, the wells were washed five times with sterile distilled water to remove all loosely attached cells. The plate was air dried for 45 min and biofilm was stained with 250 μl of 0.41% w/v crystal violet dye (AC447570500, ACROS organics) for 45 min. The dye was pipetted out from individual wells and to remove excess of dye, wells were washed five times with sterile distilled water. Plates were air dried for 45 min and thereafter, stained wells were destained with 95% ethyl alcohol. The OD₆₀₀ was measured using a micro-quant microplate spectrophotometer (Model ELx800, BioTek Instruments, Winooski, VT). The experiment was replicated four times with eight replicate wells used for each treatment.

Animal blood in each group (n=8) was obtained from abdominal aorta aseptically at 48 h after reperfusion. The levels of TNF-α, IL-1β and IL-6 in serum were assessed using ELISA kits (Jiancheng Biological Engineering Institute, Nanjing, China). Protein concentration was measured by interpolation onto absorbance curves generated by recombinant TNF-α, IL-1β or IL-6 protein standards with an ELISA reader (Model ELX800, BioTek, USA).