Dm il led inverted laboratory microscope

The preliminary screening experiments for the estimation of the silencing effectiveness of the different stem-loop constructs comprised of the transfection of insect cells followed by visual estimation by fluorescence microscopy and subsequent flow cytometry analysis. Sf9 cells were seeded to 6-well plates with a density of 9 × 10⁵ cells/well and were then pairwise co-transfected with 200 ng of the reporter plasmid pACEBac1ie1-eYFP in combination with 2 µg of one of the eight following plasmids: pACEBac1ie1amiR-1A-D or pACEBac1ie1amiR-1As-Ds. The co-transfections were done with FuGene HD transfection reagent (Promega, Madison, WI, USA) according to the manufacturer’s instructions. Forty-eight hour post-transcription, the eYFP fluorescence intensity was first evaluated using a Leica DM IL LED Inverted Laboratory Microscope and the Leica Application Suite v4.6 software (Leica Microsystems, Wetzlar, Germany). After harvesting the cells, flow cytometry analysis was carried out using a Gallios Flow Cytometer (Beckman Coulter, Vienna, Austria). For the evaluation of the raw data, the Kaluza1.2 software (Beckman Coulter) was applied. All co-transfection experiments were repeated thrice.