Microplate manager software

IL-4, IL-10, and TNF-α levels determination was done using sandwich ELISA [20 (link)]. The absorbance at 405 nm was estimated using ELISA micro-plate reader. The calculation of cytokine concentration from the standard curve utilizing Microplate Manager Software (Bio-Rad, Hercules, CA, USA) was carried out.

Immulon 2 plates (ThermoLabsystems, Pittsburgh, PA) were coated overnight at 4 °C with goat anti-mouse human adsorbed unlabeled IgM (Southern Biotech; 100 μl/well). Plates were washed, blocked with 3% BSA in PBS at 37 °C overnight, washed, and 100 μl/well of four two-fold dilutions of sera in PBS + 1% BSA were added starting at 1:2. Sample containing plates were incubated for 1 h at 37 °C and washed. Goat anti-mouse AP conjugated antibodies (Southern Biotech; 100 μl/well) were added for 1 h at 37 °C. Plates were washed and 100 µl of phosphatase substrate tablets (Sigma-Aldrich) dissolved in p-Nitrophenyl Phosphate, Disodium Salt (PNPP) buffer was added to wells. Absorbance was read at 405 nm on an xMark™ Microplate Spectrophotometer with the Microplate Manager™ Software (Bio-Rad, Hercules, CA). Standard curves were generated using serial dilutions of purified rat anti-mouse IgM (Clone II/41, BD), and the 4-parameter fit equation used to calculate sample concentrations.

Proteins (insulin, glucagon, and somatostatin) were extracted from 50 IEQs using 300 µL RIPA buffer (Sigma-Aldrich) containing protease and phosphatase inhibitor (Nacalai Tesque, Inc., Kyoto, Kyoto, Japan). Insulin content was measured using the enzyme-linked immunosorbent assay (ELISA) with the Mouse Insulin ELISA Kit (RTU) (FUJIFILM Wako Shibayagi Co., Shibukawa, Gumma, Japan). Somatostatin content was determined using the Somatostatin ELISA kit (Phoenix Pharmaceuticals, Inc., Burlingame, CA, USA). iMark Plate Reader (Bio-Rad Laboratories, Inc.) at OD450 with Microplate Manager Software (ver. 6.3, Bio-Rad Laboratories, Inc.) was used for reading sample absorbance.

The cytocompatibility of cyclodextrin formulations was evaluated on BALB/3T3 clone A31 (ATCC^® CCL-163TM) murine fibroblasts using the WST-1 test. BALB 3T3 cells were cultured in DMEM/F12 culture medium (Corning) supplemented with 10% fetal bovine serum (Hyclone) and 1% penicillin/streptomycin (Gibco). They were seeded in a 96-well plate at 1.5 × 10⁴ cells/well. To allow complete cell attachment, cells were incubated 4 h at 37 °C and 5% CO₂. Aliquots of A1 to A9 formulations, Nevanac 3 mg/mL suspension, and control (DMEM/F12) were diluted 1:50, 1:100, and 1:150 times, respectively, with complete cell culture medium to be below the IC₅₀ of nepafenac, to ensure that nepafenac was not in cytotoxic concentrations, and added to cell monolayers [45 (link)]. DMEM/F12 medium was used as control. After 24 hours of incubation with the formulations, WST-1 reagent (Roche) was added and the assay was carried out according to the manufacturer’s instructions. The absorbance was measured at 450 nm using a Model 680 microplate reader from Bio-Rad (Hercules, CA, USA) and Microplate Manager software (Version 5.2.1, BioRad, CA, USA).

STC-1 cells were maintained in RTC-coated 96-well cell culture plates and were allowed to reach 85–95% confluence. On the day of the experiment, the culture medium was replaced with DMEM containing 5.6 mM glucose and 0.1% bovine serum albumin (BSA) and the cells were then serum starved for 3 hours while equilibrated in a tissue culture incubator. The medium was then replaced with the fresh DMEM containing 5.6 mM glucose and 0.1% BSA with or without the indicated test solutions so that there were four wells per each experimental condition. STC-1 cells were exposed to these test solutions for 30 min while again being equilibrated in a cell culture incubator. Medium from each of the four wells was collected and stored at −80 °C prior to immunoassays. GLP-1 in these samples was detected using a GLP-1 Total ELISA kit (Cat. No. EZGLP1T-36K; EMD Millipore, Billerica, MA) according to the manufacturer’s instructions. O.D. values for ELISA assay samples were measured using a Benchmark Plus plate-reading spectrophotometer under the control of Microplate Manager software (Bio-Rad Laboratories, Hercules, CA). Each experiment was repeated 3 times so that the data are the average of N = 3 experiments. Data were evaluated for statistical significance by a paired t test. A p-value of <0.05 was considered to be statistically significant.

The nasal secretions were analyzed for the presence of antimicrobial peptides and proteins, IL-8 (as a measurement for inflammation) and albumin (as a measurement for vascular leakage). In addition, hCAP18/LL-37 levels were analyzed in plasma. Commercially available ELISA kits were used to detect LCN2 (Bioporto), hCAP18/LL-37 and HNP1-3 (Hycult Biotech), and IL-8 (Sanquin). The SLPI ELISA was developed in our laboratory at the Leiden University Medical Center[21 ]. The absorbance was measured at 450 nm using a Microplate reader (model 680; Bio-Rad, Hercules, CA) and Microplate Manager software (version 5.2.1, Bio-Rad). The lower limits of detection were: LCN2 10 pg/ml; SLPI 10 ng/ml; HNP1-3 150 ng/ml; LL-37 20 ng/ml and IL-8 200 pg/ml. Albumin levels were determined using nephelometry (Siemens BN Prospec) with a lower limit of detection of 17 μg/ml. Inter and intra-assay variability for all assays was < 10%.
Serum 1,25(OH)₂D3 and 25(OH)D3 were assessed at the central clinical chemistry laboratory of the LUMC. Quantification of the 25(OH)D3 concentration in the serum was done using a DiaSorin ¹²⁵I RIA Kit (DIASORIN, INC.). Quantification of the 1,25-(OH)D3 concentration in the serum was done using a DiaSorin ¹²⁵I RIA Kit (DIASORIN, INC.) preceded by extraction and column separation.