Enzyme linked immunosorbent assay plate reader

Biofilm formation test was performed according to a method described by Wakimoto et al. (17 (link)) with some modifications. Briefly, 200 µL Mueller–Hinton broth (Merck, Germany) supplemented with 0.45% glucose was added to 96-well flat-bottom microtiter polystyrene plates (Greiner, Germany) and was inoculated with 5 µL of EAEC culture grown overnight in Luria broth at 37°C with shaking. The samples were incubated overnight (18 hours) at 37°C and were visualized by staining with 0.5% crystal violet (Sigma-Aldrich, Germany) for 5 minutes after washing with water. Biofilm formation was quantified in duplicate by adding 200 µl of 95% ethanol and by using an enzyme-linked immunosorbent assay plate reader (BioTek Instruments, Winooski, VT) at 570 nm. EAEC strain 042 was used as a positive control, and E. coli HB101 was used as a negative control. Strains with OD at 570 nm of more than 0.2 were regarded as biofilm producers (biofilm-positive strains) according to a previous study (17 (link)).

A thiobarbituric acid reactive substances (TBARS; R&D Systems, Minneapolis, MN) parameter assay kit was used to measure the overall oxidative stress in 4T1 cells caused by either SPION-IL4Rα, DOX, or combined SPION-IL4Rα+DOX at different time intervals relative to controls. Briefly, cultured 4T1 cells were equilibrated overnight, treated, washed and lysed using a cell lysis buffer. Cells were incubated with an acid reagent for 15 minutes and centrifuged, after which supernatants were treated with thiobarbituric acid reagent for 2-3 hours at 45-50°C. The absorbance of the samples was measured at 532 nm using a BioTek enzyme-linked immunosorbent assay plate reader.

Cell seed preparation and viability testing were performed as described by Hassan et al.25 DMEM medium (containing 10% fetal calf serum and 1% penicillin/streptomycin solution) was used for cell culture, and cultured cells were maintained at 37°C in 5% carbon dioxide and 95% air. To avoid contamination, UV irradiation was used for the sterilization of pristine and hybrid micronanofibers. For cell seeding, the synthesized scaffolds were typically soaked in the growth medium for a few hours to aid in the appropriate attachment of cells to nanomatrix. Subsequently, fibroblasts were seeded to 60% confluency (cell count =1 × 10⁵ cells/mL) in 96-well microplates and allowed to grow for 3 days. The growth medium was changed at regular time intervals. Cells cultured without the micronanofibers served as a control.
Cell viability was measured by MTT assay. MTT reagent was prepared by strictly following the manufacturer’s instructions and was stored in the dark at 4°C until use. Briefly, following the incubation of cells with pristine and hybrid wound dressings for 1, 2, and 3 days, 40 μL of MTT reagent was added to each well which contained 160 μL growth medium and was incubated for 4 hours at 37°C. Absorbance was detected at 490 nm using an enzyme-linked immunosorbent assay plate reader (BioTek Instruments Inc, Winooski, VT, USA).

Apoptosis was assayed using Cell Death Detection ELISAPLUS Kit (Roche Diagnostics, Indianapolis, IN, USA) as we previously described (Jeong et al., 2013 (link)). This kit detects qualitatively and quantitatively the amount of cleaved DNA/ histone complexes (nucleosomes). Briefly, the cells were treated with t10, c12-CLA for 24 h and cytosolic extracts and immunoreagent were mixed and incubated for 2 h at room temperature. After washing with the incubation buffer, 100 μl of ABTS solution was added and incubated for 20 min. The absorbance was recorded at 405 nm and 490 nm in an enzyme-linked immunosorbent assay plate reader (Bio-Tek Instruments Inc., Winooski, VT, USA).

HDAC activity was determined using EpiQuik^™ HDAC Activity/Inhibition Assay Kit (Epigentek, Farmingdale, NY, USA) according to manufacturer's instructions. Briefly, Hep G2 cells were seeded in 6-well plate. After 24 h, cells were treated with 0.5 μg/ml of CPME for 48 h. After treatment, nucleus was prepared using Nuclear Extract Kit (Active Motif, Carlsbad, CA, USA). Briefly, cells were washed with ice-cold phosphate buffer saline (PBS) containing phosphatase inhibitors, resuspended with hypotonic buffer and detergent, and then centrifuged to obtain cytosolic fraction. Nuclear fractions were collected by suspending nuclear pellet with lysis buffer and centrifugation was again carried out. Biotinylated HDAC substrate was added to 8-well strip plate for 45 min. After washing the mixtures containing nuclear extract and HDAC assay buffer were added to the plate. After 1 h, the plate was washed, and the capture antibody was added for 1 h. After adding detection antibody and developing solution, the absorbance was recorded at 450 nm in an enzyme-linked immunosorbent assay plate reader (Bio-Tek Instruments Inc.) after adding stop solution. The known HDAC inhibitor trichostatin A (25 nM) (Sigma, India) was used as positive control.