Click it plus edu cell proliferation kit for imaging

EdU labeling was performed as described previously [14 (link)]. Briefly, BHK-21 cells plated on coverslips were infected with ADRV or RGV at an MOI of 0.5 and incubated at 28 °C. EdU (Invitrogen) was added to a final concentration of 10 μM at the times indicated. After continued incubation for 30 min, the cells were washed with phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde, and then processed with a Click-iT Plus EdU Cell Proliferation Kit for Imaging (Thermo Scientific, Waltham, MA, USA) according to the manufacturer’s protocols. Alexa Fluor 488-azide was used in the kit to label the EdU. In the following immunofluorescence assay, the EdU labeled cells were then incubated with antibodies against MSH2 (rabbit anti-MSH2 antibody, ABclonal), followed by the Alexa Fluor 546 conjugated Donkey anti-Rabbit IgG (Invitrogen, Waltham, MA, USA). Cell nuclis were stained with DAPI. Images were collected on a Leica TCS SP8 confocal microscope.

GSTC cells were seeded on coverslips in a 12-well plate and mock infected or infected with ADRV at an MOI of 0.5. For EdU labeling, EdU (Invitrogen, Carlsbad, CA, USA) was added to a final concentration of 10 μM at the indicated times. After a continued incubation for 30 min, the cells were washed with phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde for 20 min, and then processed with a Click-iT Plus EdU Cell Proliferation Kit for Imaging (Thermo Fisher, Waltham, USA) according to the manufacturer’s protocols as described previously [20 (link)]. For antibody staining, EdU labeled or unlabeled cells were incubated with anti-ADRV 12L or anti-ADRV 85L serum at a dilution of 1:100 as the primary antibody. After washing with PBS containing 1% BSA, the cells were incubated with secondary antibody (Alexa Fluor 546 conjugated goat anti-mouse IgG, Alexa Fluor 488 conjugated goat anti-mouse IgG, or Alexa Fluor 546 conjugated goat anti-Rabbit IgG) at a dilution of 1:1000. Hoechst 33342 was used to stain the cell nucleus. The samples were examined under a Leica TCS SP8 confocal microscope.

Nineteen- or twenty-three-hours post-infection, A549 cells were treated with EdU (Click-iT Plus EdU Cell Proliferation Kit for Imaging, Invitrogen) at a final concentration of 10 μM according to the manufacturer’s instructions. After one hour of incubation, cells were fixed with a mix of methanol/acetone 1:1 and incubated 5 minutes at -20°C. Cells were then washed once with PBS and permeabilized using the permeabilization buffer (Invitrogen) according to manufacturer’s instructions. Cells were then incubated overnight at 4°C with anti-Human APOBEC3B monoclonal antibody (5210-87-13, cat# 12397) used at a dilution of 1:250 in permeabilization buffer supplemented with 2% of goat serum (Gibco). After 2 washes in permeabilization buffer supplemented with goat serum, cells were incubated 1 hour with an anti-rabbit AlexaFluor 488 (Invitrogen). Cells were washed twice with permeabilization buffer supplemented with 2% of goat serum. EdU labelling was then performed according to manufacturer’s instructions. Cells were washed twice with permeabilization buffer supplemented with 2% of goat serum and once in PBS. Finally, slides were mounted in Fluoromount G (Invitrogen) and imaged using Zeiss LSM 900 Airyscan 2 Multiplex microscope. Images were then processed using Fiji software [54 (link)] and colocalization coefficients were calculated using Manders’ method [55 (link)].

Ovaries were directly dissected into a solution of 15 µM EdU in Schneider’s Drosophila media (500 µl, Gibco) and incubated for one hour at room temperature. These tubes were laid on their side and rocked manually, to ensure all dissected ovaries were fully submerged. Ovaries were then fixed in 3.7% paraformaldehyde in PBS for 10 min, treated with Triton in PBS (500 µl, 0.5% v/v) for 20 min, and rinsed 2x with bovine serum albumin (BSA) in PBS (500 µl, 3% w/v) for 5 min each rinse. Ovaries were exposed to the Click-iT Plus reaction cocktail (500 µl) for EdU visualization, for 45 min. The reaction cocktail was freshly prepared prior to use, with reagents from the Invitrogen Click-iT Plus EdU Cell Proliferation Kit for Imaging, including the Alexa Fluor 594 dye. Ovaries were then rinsed 3x with BSA in PBS (500 µl, 3% w/v) for 5 min each rinse.

Cell proliferation was measured by CCK-8/WST-1 or EdU assays. For EdU assays, we used the Click-iT™ Plus EdU Cell Proliferation Kit for Imaging (Invitrogen). After EdU incubation, cells were fixed with 4% paraformaldehyde and permeabilized with 0.3% Triton X-100, and EdU detection was performed according to the manufacturer’s instructions. Nuclei were counterstained with Hoechst 33342 reagent. At least 500 nuclei were counted in triplicate, and the number of BrdU-positive nuclei was recorded. For colony-formation assays, Ctrl and shRNA-infected cells were seeded in six-multiwell plates. After 2 weeks, cells were fixed with 4% PFA and stained with crystal violet. For neurospheres, 1000 cells/ml were seeded in low attachment 96-multiwell plates in DMEM with neurosphere medium as previously described^{13 (link),89 (link)}. The number of neurospheres was counted and captured images after 10–15 days.