Hybrid cell count module bz h4c

HeLa Tet-On 3G cells were cultured in DMEM (WAKO) with10% Fetal bovine serum medium. For transfection of the expression vectors, ratiometric fluorescence reporter in pTRE-Tight-BI with or without SECIS-like fragment, 5×10⁴ cells were seeded in 24-well plates, or 2.5x10⁴ cells were seeded in 48-well plates (Thermo Fisher). Seven hours after transfection, the culture medium containing transfection reagents was changed to fresh medium containing 10 ng/mL doxycycline. Twenty-four hours after transfection, Images of Venus (50ms) and tdTomato (5ms) were obtained from three fields of view for each well by the KEYENCE BZ-X810. Nuclei with Venus and tdTomato were automatically selected and the sum of the Venus and tdTomato signals in each well was calculated from the Venus and tdTomato signal intensities of each cell in the well by Hybrid Cell Count Module BZ-H4C (KEYENCE). The relative sum of the tdTomato signal intensity to the sum of the Venus signal intensity in each well was calculated as the translational activity and relative values are shown in the graph. Quantification of Venus and tdTomato fluorescence signal from single cells were automatically obtained and quantified using the Hybrid Cell Count Module BZ-H4C (KEYENCE).

Venus and tdTomato signal intensities were obtained for 20 and 5 ms, respectively. Images were obtained from eight fields of view for each well, and nuclei with Venus signals were selected by the NEW Opera Phenix HCS System (PerkinElmer). The sum of the Venus and tdTomato signals in each well was calculated from the Venus and tdTomato signal intensities of each cell in the well. The relative sum of the Venus signal intensity to the sum of the tdTomato signal intensity in each well was calculated as the pri-miRNA processing activity and relative values are shown in the graph. Alternatively, the cell images were obtained by KEYENCE BZ-X810. Quantification of Venus and tdTomato fluorescence signal from single cells were automatically quantified using the Hybrid Cell Count Module BZ-H4C (KEYENCE). The slope of the Venus and tdTomato signals as the pri-miRNA processing activity is shown in the graph.