Sunrise elisa reader

Quantification of the identified protein targets from the iTRAQ-based quantitative proteomic analysis was further validated by ELISA. Serum levels of catalase (CAT), peroxiredoxin-2 (PRDX2), carbonic anhydrase 1 (CA1), and carbonic anhydrase 2 (CA2) were determined using ELISA kits (CAT: #ab171572, Abcam, Cambridge, UK; PRDX2: #EK1528, Boster, Pleasanton, CA, USA; CA1: #OKEH01283, Aviva System Biology, San Diego, CA, USA; CA2: #ab222881 Abcam, Cambridge, UK) and a TECAN Sunrise ELISA Reader (TECAN, Lake Zürich, Switzerland).

Cells (5 × 10³ per well in 96 well plates or 2 × 10⁵ cells per 6‐cm dish) were treated with cisplatin (2 μg/mL), LBH589 (100 nmol/L), rapamycin (100 nmol/L) or a combination of two drugs for 48 hours. Cell viabilities were determined by Celltiter‐Glo Luminescent Cell Viability Assay (Promega) according to the manufacturer's instructions. At the time of assay, CellTiter‐Glo reagent was added and mixed for 2 minutes on an orbital shaker. Luminescent signals were obtained using TECAN sunrise ELISA reader (Tecan, Trading AG) after 10 minutes of incubation.

Cytotoxicity of HH-F3 was evaluated by measuring LDH activity released in the media after the exposure according to the manufacturer’s instructions (Pierce, USA). Cells without any treatment served as the spontaneously LDH activity control. Following treatment, supernatants from cell without treatment, cells with treatments, and cells treated with lysis buffer were collected and incubated with LDH assay solution accordingly. The optical density values were analyzed at 490 nm by subtracting the reference value at 680 nm using a TECAN sunrise ELISA reader (Tecan Trading AG, Switzerland). LDH positive controls provided in the kit was included in the assay to confirm the success of the assay. The results were expressed as a percentage of treated sample LDH activity (LDH release obtained from treated cells subtracting to spontaneous LDH release obtained from untreated cells) to the total LDH activity (maximal LDH release obtained from lysed cells subtracting to spontaneous LDH release obtained from untreated cells).

Serum was obtained after centrifugation of the blood for 10 min at 10,000× g and stored at −20 °C until further use. The PR8 H1N1 antigen-specific IgG antibody responses induced by influenza PR8 challenge were evaluated by ELISA. PR8 H1N1 hemagglutinin protein (Sino Biological Inc., Beijing, China), 1 µg/mL, was coated onto MaxiSorp plates (Nunc, Hillerød, Denmark) overnight at 4 °C. Serum was added at 10-fold serial dilutions and incubated for 2 h at room temperature (rt), followed by incubation with HRP-conjugated anti-mouse total IgG antibodies (AH diagnostics, Tilst, Denmark) for 1 h at rt. The signal was detected by TMB (Kem-En-Tec, Taastrup, Denmark) and the reaction stopped with 0.2 M of H₂SO₄, followed by analysis on a TECAN Sunrise™ ELISA reader (Tecan Trading AG, Männedorf, Switzerland) at 450 nm with 620 nm correction.

ELISA was performed to measure the titers of Ag-specific IgG1 and IgG2a antibodies in serum. Nunc 96-well MaxiSorp plates (Thermo Fischer Scientific) were coated with 100 µl 1 µg/ml unmodified LYS in carbonate buffer (pH 9.6) (SSI Diagnostica, Hillerød, Denmark). The plates were incubated at 4 °C overnight and washed with 0.2% Tween (Merck) in PBS (pH 7.2, Gibco Life Technologies™). The plates were blocked for 1.5 h with 2% BSA in PBS. To give a 10-fold dilution curve, 1% BSA in PBS was added to each well and the serum was added. The plates were incubated at RT for 2 h. After washing the plates, they were incubated for 1 h with either horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG1 (Invitrogen, Carlsbad, CA, USA) diluted 1:20,000 in 1% BSA in PBS or HRP-conjugated rabbit anti-mouse IgG2a (Invitrogen) diluted 1:5,000 in 1% BSA in PBS. The plates were developed with 100 μl/well 3,3′,5,5′-tetramethylbenzidine substrate (Kem-En-Tec Diagnostics, Taastrup, Denmark) for 10 min. The reaction was stopped by adding 0.2 M sulfuric acid (VWR, Radnor, PA, USA), and the plates were analyzed using a TECAN Sunrise™ ELISA reader (Tecan Trading) at OD₄₅₀ nm corrected at OD₆₂₀.

The protein concentrations of MMP-9 in the plasma were measured by using the commercially available DuoSet ELISA development kits (R&D Systems Inc., McKinley Place N.E., Minneapolis, USA) according to the manufacturer's protocol (MMP9 catalog number DY911). The absorbance of the color at 450 nm was recorded using a TECAN Sunrise ELISA Reader (Tecan Group Ltd., Männedorf, Switzerland).