In cell analyzer 6000 microscope

Cell transfection assays were performed by electroporation using the Neon Transfection System. Cells were electroporated with pmTFP1-NKEF at different plasmid concentrations (for RBCs: 0.5 μg, 1 μg, and 2 μg; for EPC: 50 ng, 100 ng, and 150 ng) or with pmTFP1 (RBCs: 2 µg and EPC: 250 ng) at 1600 V, 30 ms, 1 pulse. EPC cells were chosen for this assay because they are highly susceptible to gene transfection [35 (link)]. The protein expression of NKEF in transfected cells was monitored at different time points (for RBCs: 1, 3, and 6 days post-transfection; for EPC: 1, 2, and 3 days post-transfection) by evaluating the fluorescence emitted by the reporter protein TFP1 bound to NKEF protein (NKEF-mTFP1) using the IN Cell Analyzer 6000 microscope (GE Healthcare, Little Chalfont, UK) and quantified by flow cytometry using the FACSCanto II flow cytometer (BD Biosciences).
Moreover, cells were resuspended in TRK lysis buffer for RNA extraction to evaluate the immune response triggered by NKEF overexpression by means of RT-qPCR.

Cells were cultured in CellCarrier-96 plates (PerkinElmer). After the appropriate length of treatment with Dox ± inhibitors, cells were fixed with 4% formaldehyde/PBS (v/v) and permeabilised with 0.2% Triton X-100 for 10 min. For analysis of GFP intensity, cells were stained with DAPI at a concentration of 1 µg/ml for 10 min. For detection of p-ERK1/2, p-MEK1/2, total ERK1/2 or total MEK1/2, cells were blocked for 1 h with 2.5% goat serum (v/v) in 2% BSA/PBS (v/v) at room temperature. Cells were then incubated with primary antibody diluted in 2% BSA/PBS overnight at 4°C. Background control wells were treated with 2% BSA/PBS, without the addition of primary antibody. Cells were washed three times with PBS and then incubated with Alexa Fluor secondary antibodies (1:500) and DAPI (1 µg/ml) in 2% BSA/PBS for 1 h. Cells were washed with PBS before imaging. Cells were imaged using an INCELL Analyzer 6000 Microscope (GE Healthcare), imaging six fields per well. Image analysis to determine the intensity of GFP or fluorescent signal was performed using INCELL Analyzer software.