The SARS-CoV-2 Neutralizing Antibodies Detection Kit (#AG-48B-0002-KI01, AdipoGen Life Science, San Diego, CA, USA) was procured for the analysis. This kit utilizes antigens derived from SARS-CoV-2 (Wuhan-Hu-1 strain). Initially, the RBD protein, a key component of the kit, was pre-coated onto a 96-well plate. For the assay, the 100 μL/well of either 10-fold diluted serum or undiluted bronchoalveolar lavage (BAL) samples were added and incubated at 37 °C for 1 h. This step was followed by the addition of 100 μL/well of ACE2 (human)-HRP conjugate, which was also incubated for 1 h at 37 °C. Subsequently, 100 μL/well of the 3,3′,5,5′-tetramethylbenzidine (TMB) solution was introduced and incubated for 10 min in a dark environment. The reaction was halted by adding 100 μL/well of stop solution. Absorbance was then measured at 450 nm using a microplate reader (MTP-880Lab; Corona Electric, Hitachinaka, Japan). The quantification of neutralizing antibodies against SARS-CoV-2 in the serum/BAL samples was determined by calculating the percentage inhibition using the formula: (1 − (OD of the sample/OD of Negative Control)) × 100. The negative control comprised human serum diluted 1/10, confirmed to be negative for SARS-CoV-2 antibodies and screened for viral markers.
Mtp 880lab
Manufactured by Corona Electric
Sourced in Japan
The MTP-880Lab is a multifunctional laboratory equipment designed for various scientific applications. It is a compact and versatile device that offers essential functionalities for research and testing purposes. The core function of the MTP-880Lab is to provide reliable and accurate measurements and analysis across a range of parameters, supporting a wide variety of laboratory tasks.
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7 protocols using mtp 880lab
1
SARS-CoV-2 Neutralizing Antibody Detection
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Cytotoxicity of Carboxyl Magnetic Microspheres
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The cytotoxicity of the carboxyl magnetic microspheres was investigated using a CCK-8 method in vitro. The 96-well plates were seeded with a suspension of 5000 human pulmonary epithelial cells for 24 h to allow the cells to adhere. Then serial dilutions of carboxyl magnetic microspheres solution, the supernatant and medium alone (control) were added into the wells. After incubation at 37 °C for 24 h in an atmosphere of 5% CO2, 10 μL CCK-8 solution was added to each well and the cells were incubated for another three hours. Absorbance at 450 nm was determined using a microplate reader using a microplate reader (MTP-880 Lab, Corona Electric, Ibaraki, Japan). Cytotoxicity was expressed as a percentage of viable cells compared with untreated control ones.
3
CCK-8 Assay for Cell Viability Evaluation
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RAW264 cells were seeded in 96-well plates at a density of 5000 cells/well (in 180 μL of medium). After incubation for 24 h in a humidified incubator with 5% CO2 at 37 °C, 20 μL of ε-PLL solution dissolved in 1× D-PBS was added to the culture medium. After another 24 h of incubation, 10 μL of CCK-8 reagent was added to the medium, which was further incubated for 4 h with 5% CO2 at 37 °C. Absorbance at 450 nm was measured using a microplate reader (MTP-880 Lab; Corona Electric, Ibaraki, Japan). The cell viability was calculated using the formula below. The medium without cells was used as a blank. Cells in which only 1× D-PBS buffer was added were used as controls.
4
Curcumin-Loaded Liposome Characterization
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Yield percentage was calculated based on the dry solid weight of liposomes compared to their initial weight. Amount of Cur entrapped in the liposomes was determined by a spectrophotometric method. To do this, the liposomes were mixed in ethanol at 2.5% vol to disrupt the structure and dissolve Cur. The absorbance of the ethanolic solutions at 415 nm was collected using a microplate reader (MTP-880Lab, Corona Electric, Japan). Loading capacity (LD) and encapsulation efficiency (EE) were calculated using the following equations: where Cur abs is the amount of Cur in mg calculated from the ab- sorbance value, W lip is the solid weight of liposomes after preparation, and Cur cal is the theoretical amount of Cur calculated based on the W lip .
5
SARS-CoV-2 Spike Protein ELISA
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RBD protein (Abcam, recombinant human coronavirus SARS-CoV-2 Spike Glycoprotein RBD [Active] 50 µg, #ab273065, Cambridge, UK) or S1 protein (Sino Biologicals, SARS-CoV-2 Spike S1-His Recombinant Protein [HPLC-verified], #40591-V08H) was diluted with a coating solution (0.1 mM NaHCO3, pH 9.6) to a concentration of 2 µg/mL. Microtiter plates (430341, Nunc C96 Maxisorp; Thermo Fisher Scientific, Waltham, MA, USA) were coated for 2 h at 4 °C with each diluted antigen. Subsequently, 200 µL/well of blocking solution was added and incubated overnight at 37 °C or for 2 h or four times. A 1000-fold dilution of mouse serum or BAL fluid was used as the primary antibodies and added to the plates (100 μL/well) and incubated overnight at 4 °C. Peroxidase-labeled anti-mouse IgG (Sigma-Aldrich, anti-mouse IgG [whole molecule] produced in goat-affinity isolated, buffered aqueous solution, #A4416, St. Louis, MO, USA) and anti-mouse IgA (Abcam, Waltham, MA, USA, goat anti-mouse IgA alpha chain [HRP], #ab97235) were applied at 1:60,000 for 1 h at 37 °C. After six washes, the plates were incubated with 2,2′-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) (A3219; Sigma-Aldrich) at room temperature for 30 min. Next, 0.5 M H2SO4 at 100 µL was added per well, and the optical density (OD) was measured at 450 nm using a microplate reader (MTP-880Lab; Corona Electric, Hitachinaka, Japan).
6
SARS-CoV-2 Neutralizing Antibody Assay
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Neutralizing antibodies were purchased from AdipoGen Life Science (SARS-CoV-2 Neutralization Antibodies Detection Kit, #AG-48B-0002-KI01, San Diego, CA, USA). The antigens were obtained from SARS-CoV-2 (Wuhan-Hu-1). The RBD protein was preliminarily immobilized on the 96-well plate provided in the kit, and 100 µL/well of a 10-fold diluted serum or undiluted BAL samples were added and incubated at 37 °C for 1 h. Subsequently, 100 µL/well of ACE2 (human)-HRP was added and reacted for 1 h at 37 °C; a total of 100 µL/well of 3,3′,5,5′-tetramethylbenzidine (TMB) solution was added and incubated for 10 min in the dark before adding 100 µL/well of stop solution and measuring the absorbance at OD450 nm using a microplate reader (MTP-880Lab; Corona Electric, Hitachinaka, Japan).
7
NIPAM-hemin Copolymer Cytotoxicity Assay
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The 293XL/null cells and RAW 264 cells were seeded in 96-well plates at a density of 5,000 cells/well and incubated at 37°C in a humidified incubator containing 5% CO2 for 24 h. The medium was then replaced with fresh high-glucose DMEM supplemented with 10% (v/v) heat-inactivated FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin. NIPAM-hemin copolymer was added to the medium at a final concentration of 0, 0.25, 0.5, 0.75, 1.0, or 2.0 mg/mL. After an additional 24 or 48 h incubation, the medium was replaced with 100 μL fresh medium, and 10 μL CCK-8 solution was added to each well, followed by incubation for 1 or 3 h at 37°C in a humidified incubator containing 5% CO2. The absorbance at 450 nm (A450) of each well was measured using a microplate reader (MTP-880Lab; Corona Electric, Ibaraki, Japan). An in vitro cytotoxicity assay for poly-NIPAM was also conducted using the same procedure described above.
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