Spectramax abs

To determine antigen‐specific IgG titers, recombinant spike‐WT, spike‐BA.5, spike‐BF.7, spike‐BQ.1.1, spike‐XBB, or spike‐XBB.1.5 proteins were used to coat 96‐well plates (NUNCMaxiSorp, Thermo Fisher Scientific) in carbonate coating buffer (50 mM, pH 9.6) at 1 µg/mL at 4°C overnight. After three washes with PBS containing 0.1% Tween‐20 (PBST), the precoated plates were blocked with 200 µL/well of 1% bovine serum albumin for 1 h at room temperature. The immune sera were thawed slowly at 4°C, serially diluted, added to the plates, and incubated at 37°C for 1 h. After another three washes with PBST, horseradish peroxidase‐conjugated anti‐mouse IgG antibodies (Invitrogen, 1:10,000) were added to the plates (100 µL/well) and incubated for 1 h at 37°C. Then, the plates were washed five times before the addition of 3,3′,5,5′‐tetramethyl biphenyl diamine (100 µL/well) for color development. After 10–15 min, 100 µL/well of ELISA stop solution (Beyotime, China) was added to stop the reaction. Finally, a microplate reader (Spectramax ABS, Molecular Devices) with SoftMax Pro 7.1 software was applied to read the absorbance value.

Blood was collected immediately at euthanasia, left at RT for 20 minutes and centrifuged at 1100 x g for 30 minutes to separate the serum. The serum was stored in a new vial at −80 °C until used. Concentration of progesterone in blood serum was measured with a competitive ELISA for mouse progesterone (Crystal Chem). Optical density of each replicate was measured with an absorbance reader (SpectraMax ABS by Molecular Devices) at 450/630 nm wavelength. Progesterone concentration was determined in comparison to standards using (SoftMax Pro 7.1 software).

Escherichia coli BCRC 13B0198 were cultured at the mid-log phase and diluted to 10⁴ CFU/ml, and then treated with peptide alone at 1 × MIC or combination with vancomycin (both at 0.5 × MIC) at 37°C for 6 h. The cells were filtered by using a pyrogen-free 0.2 μm pore filter (Acrodisc, Pall Corporation, United States) and the endotoxin level was determined by limulus amebocyte lysate (LAL) PYROCHROME^® test (Associates of Cape Cod, United States). The kinetic turbidity was analyzed by using a microplate reader (SpectraMax ABS, Molecular Devices, San Jose, CA, United States). The experiments were repeated three times independently.

At the end of the culture, cells were fixed with 4% paraformaldehyde at 4 °C for 1 h. Lipid droplets were stained with 3 mg/mL Oil Red O for 1 h at room temperature. After washing, the dye incorporated into the cells was re-extracted with 2-propanol, and the absorbance of the extracts at 540 nm was measured using SpectraMax ABS (Molecular Devices, San Jose, CA, USA). Lipid accumulation was expressed as relative absorbance compared to the control value.

To determine the toxicity of the ZnO NPs, HK2 cells in RPMI 1640 medium supplemented with 10% FBS were seeded into a 96-well culture plate (1 × 10⁴ cells/200 µL/well) in the presence of increasing concentrations of ZnO NPs (0, 1, 2, 5, and 10 µg/mL). The HK2 cells were incubated for 2 days at 37 °C in the presence of CO₂. At the end of the incubation period, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was conducted to determine the viability of the HK2 cell. The cells following exposure to ZnO NPs for 24 h were incubated with 2 mg/mL MTT at 37 °C for 3 h in the dark. After removing the previous incubated medium, dimethyl sulfoxide (DMSO) was added to dissolve formazan transformed by live cells. Absorbance was measured at 540 nm by a microplate reader (SpectraMax^® ABS, Molecular Devices, San Jose, CA, USA).

On day 1, PC3 and RWPE-1 cells were seeded in 96-well culture plates (#3904, Corning, USA) at a certain cell density per well (e.g., 10,000 cells/well) to maintain uniformity throughout the cell viability assay. After cell attachment occurred on day 2, cells were treated with strictinin in fresh media at 1,000 µmol/L, 500 µmol/L, 250 µmol/L, 125 µmol/L, 62.5 µmol/L, and 31.25 µmol/L. These concentrations were achieved through serial dilution. After 24–72 h post-treatment, the strictinin-containing media was aspirated and fresh media was introduced along with methyl thiazolyl tetrazolium (MTT) dye. Cells were incubated in MTT dye for 2–4 h at 37℃ followed by adding DMSO and reading the plates (Spectramax ABS, Molecular Devices, USA) at an absorbance value of 540 nm. Cell viability was calculated as the ratio of absorbance readings from treated wells to non-treatment or vehicle control wells.