Wallac 1420 victor 3v

Cells were subcultured in 12-well plates at a density 5 x 10⁴ cells/well one day before transfection. To investigate the effect of PIN1 silencing on EPO-HRE reporter activity, cells were transfected with PIN1 siRNA or mock siRNA for 72 h at 37°C. The cells were transfected with plasmids including luciferase-linked reporter gene (EPO-HRE luc) and β-galactosidase (β-gal) expression vector. After 24 h transfection, cells were lysed, and luciferase activities were measured. The β-gal activity was used to normalize transfection efficiency.
Bioluminescence image was obtained using a luciferase assay kit (Applied Biosystems, Carlsbad, CA, USA). The plates were washed twice with PBS, and then lysis solution was added to each well. Cell lysates were transferred to a 96 well microplate and luciferase activities were measured by a Wallac 1420 VICTOR3 V (PerkinElmer Life and Analytical Sciences, Shelton, CT, USA) luminometer.

A fluorescence assay with Vybrant™ DiO Cell-Labeling Solution (Invitrogen; Thermo Fisher Scientific, Waltham, MA, USA) was performed in order to confirm OMV quantification of ULS153 and ULS153 ΔDlm strains. For each sample, 15 µL of purified OMVs were suspended in 85 µL of PBS and the mixture was labeled with 1% Vybrant™ DiO by incubation for 20 min at 37 °C [39 (link)]. Free dye was removed by two washes with 15 mL of PBS using the 100K Amicon^® Ultra-15 centrifugal filter device. Labeled OMVs were collected and stored at −20 °C. The concentration of OMVs was determined by measuring fluorescence at 485-nm excitation and 535-nm emission wavelengths with a Wallac 1420 Victor³V microplate reader (Perkin-Elmer, Waltham, MA, USA).