Spectramax i3 microplate reader

According to the manufacturer’s instructions, the amount of Granzyme B produced by activated T cells in cell coculture was determined using culture supernatants that were gathered, spun free of cells, and frozen at -20 °C. The wells were blocked for 1 h at room temperature using PBS, 0.05% Tween-20, and 0.1% bovine serum albumin (BSA) after two PBS washes. After adding the samples for 2 h at room temperature, wells were washed and further 1 g/mL Granzyme B-biotin was added for 1 h. Wells were washed and were incubated in 1 g/mL of streptavidin-HRP for 1 h. Wells were washed following the addition of streptavidin-HRP, and then TMB substrate was added for up to 20 min (Biolegend). After adding the TMB stop solution to terminate color development, relative absorbance was measured at 450 nm using a SpectraMax i3 microplate reader (Molecular Devices, San Jose, CA, USA).

All rose varieties used in this study were planted and collected from the National Rose DUS examination station, which is responsible for the DUS testing for rose varieties (Kunming, Yunnan). Fresh leaves were collected from healthy and strong plants, wrapped and stored on dry ice (− 70 °C) until analysis; a list of tested rose varieties is shown in Table S5. Rosa ‘Margo Koster’ was used to perform next-generation sequencing to construct the whole cp genome, and other rose varieties were used to test and validate the polymorphism of molecular markers. Genomic DNA was isolated using a DNA extraction Kit (DP-305, Tiangen Biotech, Beijing CO. LTD); agarose gel electrophoresis and a one-drop spectrophotometer were used to detect DNA integrity and quality (Spectramax I3 Microplate Reader, Molecular Devices, USA).

Cell viability was measured using the CCK-8 (cell counting kit-8, Dojindo, Gaithersburg, MD) as previously described [32 (link)]. Her2/CT26 cells were seeded at a concentration of 2×10⁵ cells/well in 96-well plates and incubated for 24 h at 37°C with 5% CO₂. The next day, cells were treated with indicated concentration of TSA. After incubation for 6 h and 24 h, Her2/CT26 cells were washed with PBS, 10 μl CCK-8 solution were added to each well and plates were incubated for another 4 h at 37°C with 5% CO₂. Cell viability was determined by reading absorbance at 450 nm using a SpectraMax I3 microplate reader (Molecular Devices, Palo Alto, CA) with a reference absorbance at 620 nm to correct for nonspecific background values.

Antiviral activity was analyzed using the SRB method to analyze cytopathic effect (CPE) reduction, as previously reported [18 (link)]. Briefly, A549 cells were seeded in a 96-well plate at a density of 3 × 10⁴ cells per well and incubated for 24 h. Following this, 1 × 10³ pfu/90 µL of virus suspension containing TPCK-trypsin (1 µg/mL) was added to each well, along with the indicated concentration of chrysin. The cells were incubated at 37 °C in 5% CO₂ for 3 days until an appropriate CPE was achieved. After 3 days, the A549 cells were washed with PBS and incubated for 30 min at −20 °C after the addition of ice-cold acetone (70%). After removing the acetone, the plate was dried for 30 min in a drying oven, after which, 0.4% (w/v) SRB in 1% acetic acid solution was added to each well and incubated for 30 min at 20 °C. The SRB solution was then removed and the plates were washed with 1% acetic acid before oven-drying. After drying for 1 day, SRB was solubilized with a 10 mM unbuffered Tris-based solution, and the absorbance was measured at 540 nm using a SpectraMax i3 microplate reader (Molecular Devices, Palo Alto, CA, USA) with a reference absorbance of 620 nm. The antiviral activity of each test compound in A/PR/8 virus-infected cells was calculated as a percentage of the corresponding untreated control.

The effects of MFN and IR on cell viability were determined using the CCK-8 colorimetric assay (Dojindo, Mashiki-machi, Japan). Hepa1-6 cells were seeded in 96-well plates, at a density of 5 × 10³ cells per well, for 12 h. After incubation with different doses of MFN, ranging from 0 to 50 μg/mL, for 24, 48, and 72 h, 10 μL of the CCK-8 solution was added to the wells and the plates were then incubated for another 2 h. The absorbance at 450 nm was measured using a SpectraMAX i3 microplate reader (Molecular Devices, San Jose, CA, USA). Relative cell viability was expressed as a percentage of the viability of the untreated control cells.