Click it edu labeling kit for microscopy

As described previously [9 (link),10 (link)], WI-38 cells were grown until 100% confluent on LabTek II 4-chamber slides (Thermo-Fisher, Waltham, MA, USA). After becoming fully confluent, cells were incubated for a further 72 h to achieve growth arrest. Infections were carried out as described above with a multiplicity of infection (MOI) of 100 for dl309 [14 (link)], or dl1101 through dl1109 [15 (link)]. One hour prior to fixation, cells were pulsed with EdU for 1 h as per manufacturer’s specifications using the Click-It EdU labeling kit for microscopy (Life Technologies, Carlsbad, CA, USA). After EdU labeling, cells were fixed in 3.7% formaldehyde, stained for EdU using the Click-It kit with AlexaFluor 488 and labelled for E1A using M58 monoclonal antibody and AlexaFluor 594 conjugated secondary anti-mouse antibody (Jackson ImmunoResearch). Cells were imaged using Molecular Devices (San Jose, CA, USA) ImageXpress Micro 4 high content imager and automatically analyzed using the MetaXpress software.

IMR-90 cells were grown until 100% confluent on LabTek II 4-chamber slides (Thermo-Fisher). After becoming fully confluent, cells were incubated for a further 72 hours to achieve growth arrest. Infections were carried out as described above with m.o.i. of 5 for dl309, and m.o.i. of 10 for dl520 and pm975. One hour prior to fixation, cells were pulsed with EdU for 1 hour as per manufacturer’s specifications using the Click-It EdU labeling kit for microscopy (Life Technologies). After EdU labeling, cells were fixed in 3.7% formaldehyde, stained for EdU using the Click-It kit with AlexaFluor 488, and labelled for E1A using M73 monoclonal antibody and AlexaFluor-594 conjugated secondary antibody (Jackson Immunoresearch). Cells were visualized using LSM700 laser confocal microscope and ZEN software suite. Quantification was carried out using ImageJ software.

As described [2 (link)], WI-38 cells were grown until 100% confluent on LabTek II 4-chamber slides (Thermo-Fisher, Waltham, MA, USA). After becoming fully confluent, cells were incubated for a further 72 h to achieve growth arrest. Infections were carried out as described above with a multiplicity of infection (MOI) of 100 for dl309 [16 (link)], or dl311 [16 (link),17 (link)], or dl1116 [18 (link)], or dl1132 [19 (link)], or dl1133 [5 (link)], dl1134 [5 (link)], or dl1135 [5 (link)], or dl1136 [5 (link)]. One hour prior to fixation, cells were pulsed with 5-ethynyl-2´-deoxyuridine (EdU) for 1 h as per manufacturer’s specifications using the Click-It EdU labeling kit for microscopy (Life Technologies, Carlsbad, CA, USA). After EdU labeling, cells were fixed in 3.7% formaldehyde, stained for EdU using the Click-It kit with AlexaFluor 488 and labelled for E1A using M58 monoclonal antibody and AlexaFluor 594 conjugated secondary anti-mouse antibody (Jackson ImmunoResearch). Cells were visualized using LSM700 laser confocal microscope (Carl Zeiss AG, Oberkochen, Germany) and ZEN software suite (Carl Zeiss AG, Oberkochen, Germany).