Nuclear id red dna stain

For RNA-Seq, UAS-expression of UAS-klu or klu^RNAi was induced using esg-Gal4^ts, UAS-GFP for 2 days, followed by 16 h of Ecc15 infection to stimulate midgut turnover. We dissected 100 midguts/genotype in triplicate and for each sample 20,000–40,000 cells were sorted into RNAse-free 1× PBS with 5 mM EDTA. RNA was isolated using the Arcturus PicoPure™ RNA Isolation Kit. Subsequently, the entire amount of isolated RNA was used as input for RNA-amplification using the Arcturus™ RiboAmp™ HS PLUS Kit. Two hundred nanograms of amplified aRNA was used as input for RNA-Seq library preparation using the TruSeq Stranded mRNA Library Prep Kit (Illumina) and samples were subsequently sequenced as 50 bp single-end on an Illumina HiSeq2500. For the FACS analysis experiment with DNA staining, we dissected 60–70 midguts/genotype under the same conditions and used NuclearID Red DNA Stain (ENZ-52406, Enzo Life Sciences) for DNA content analysis. FACS-plots were generated with FlowJo v10.

Apoptosis assays were done with the IncuCyte ZOOM. To measure apoptosis, the CellPlayer 96-Well Kinetic Caspase-3/7 reagent containing DEVD-NucViewTM488 (Essen Bioscience) at a concentration of 2 μM was added at the same time as the drug. Total numbers of apoptotic cells were counted in the green channel (488 nm). After 96 h, cells were incubated for 30 minutes with 4 µM of Nuclear-ID Red DNA stain (Enzo Life Sciences, Farmingdale, NY, USA), and total cell count was measured in the red channel (566 nm). The percentage of apoptotic cells per well was calculated as the number of apoptotic cells relative to the total number of nuclei.

Cells were collected from 6 cm plates, at about 80–90% confluence, by using trypsinization. Cells were washed with cold PBS and fixed with 95% cold ethanol for 10 min at –20°C. Cells were stained with anti-RAD51 antibody (ab1837, Abcam, 1:100) and Alexa Fluor 488 goat anti-mouse (A11001, Thermo Fisher Scientific). After washing, cells were resuspended in 200 µL PBS and NUCLEAR-ID Red DNA stain (ENZ-52406, Enzo Life Science), and incubated in the dark at room temperature for 30 min. Samples were analyzed by a BD Accuri C6 Plus Flow Cytometer.

Neutrophils were resuspended at 2 × 10⁶ cells/mL and incubated with NUCLEAR-ID^® red DNA stain (1 µL per 1.5 × 10⁶ cell suspension, Enzo Life Sciences, Farmingdale, NY, USA) for 5 min in the dark. Cells were washed 3 times in RPMI medium and spun down at 2400 g for 5 min. Stained cells were then plated at 20,000 cells per 100 µL in a 96-well poly-l-lysine coated flat bottom plate and incubated for 20 min to settle. Cells were then stimulated or not with PMA or ionomycin at given concentrations in a mixture with 2 µM SYTOX^® Green DNA dye (Thermo Fisher, Cat No. S7020, Waltham, MA, USA) and were placed in the Incucyte^® ZOOM platform contained within an incubator at 37 °C and 5% CO₂.

cDNAs encoding untagged human or mouse NINJ1 were cloned into pcDNA3.1 Zeo(+). HEK293T cells (2.6 × 10⁴) were transfected with 50 ng plasmid plus 0.16 μl Lipofectamine 2000 per well in 96-well plates in the presence of 20 or 200 μg ml⁻¹ of isotype control antibody, anti-NINJ1 clone D1 (Genentech), or anti-NINJ1 clone D1 Fab (Genentech). YOYO-1 dye (Thermo Fisher Scientific) was added at a final concentration of 200 nM at the time of transfection. IncuCyte S3 images were scanned in the green channel every hour for at least 16 h and at 10× magnification. Nuclear-ID Red DNA stain (Enzo Life Sciences) was added after the last time point and scanned in the red channel. IncuCyte S3 2019A software was used to determine the total number of YOYO-1⁺ cells and Nuclear-ID⁺ cells (total cells). The percentage of YOYO-1⁺ cells was calculated as the number of YOYO-1⁺ cells divided by the total number of Nuclear-ID⁺ cells.