qPCR reactions were performed in triplicates in a final volume of 10 μl with a cDNA amount equivalent to 10 ng of total RNA, 500 nM of each primer and 5 μl of SsoFast EvaGreen SuperMix (Bio-Rad, CN 172-5204), according to the manufacturer’s instructions. For all the genes we carried out an initial denaturation of 30’’ × 95°C followed by 40 two-step cycles (5’’ × 95°C + 5’’ × 60°C). We also included a melting curve from 60°C to 95°C (0.5°C/seg) at
the end of the program to verify the specificity of the PCR. Fluorescence was acquired during both the 60°C and melting steps. Reactions were set up robotically, with an Eppendorf pipetting robot (epMotion 5075). qPCR instrument LY2606368 ic50 was a CFX384 Real Time System C1000 Thermal Cycler (Bio-Rad). No Template Control (NTC) amplifications were always either negative or delayed more than 5 cycles with respect to the experimental samples. In order to estimate the individual efficiency of each primer pair and to validate a quantitative range for each assay we performed a qPCR over a six-point ¼ dilution curve made from a “pool” cDNA sample (cDNA input range equivalent to 50-0.05 ng total RNA). The quantification cycles (Cqs) of the experimental samples were within the ranges
validated by the dilution curves. Flow cytometry analysis To perform FACS analysis, HOG cells were dissociated by incubation for 1 minute in 0.05% trypsin/0.1% EDTA (Invitrogen) at room temperature and washed and fixed in 4% paraformaldehyde for 15 minutes. Then, cells were rinsed and resuspended in PBS. Cells were analyzed using a FACSCalibur CYT387 mouse Flow Cytometer (BD Biosciences). Immunofluorescence microscopy Cells grown on glass coverslips were fixed in 4% paraformaldehyde for 20 min, rinsed with PBS and treated with 20 mM glycine for 5 min to quench aldehyde groups. Cells were then permeabilized with 0.2% Triton X-100, rinsed and incubated for 30 min with 3% bovine serum albumin in PBS with 10% human serum, to block the HSV-1-induced IgG Fc receptors. For double and triple-labeled immunofluorescence analysis, cells were incubated for 1 hr at room temperature
with the appropriate primary antibodies, rinsed several Branched chain aminotransferase times and incubated at room temperature for 30 min with the relevant fluorescent secondary antibodies. Antibodies were incubated in the presence of 10% human serum. Controls to assess labeling specificity included incubations with control primary antibodies or omission of the primary antibodies. After thorough washing, coverslips were mounted in Mowiol. Images were obtained using an LSM510 META system (Carl Zeiss) coupled to an inverted Axiovert 200 microscope. Quantification of colocalization, was performed using M1 and M2 Manders coefficients . We calculated Manders Semaxanib overlap coefficients selecting regions of interest corresponding to the areas where the colocalization seemed to be high, that is, areas in yellow, magenta and cyan.