Hoescht 33342

After red blood cells lysis, blood cells were cytospun onto SuperFrost Plus slides. Samples were fixed in 10% formalin (Sigma–Aldrich) and then stained for CD14 (13-0149-82, Invitrogen) overnight and with the appropriate secondary antibody (Streptavidin AF 647, S32357, ThermoFisher Scientifc). Samples were then permeabilised and stained for IRF5 (10547-1-AP, Proteintech) and OXPHOS (MS604, Abcam) with the appropriate secondary antibodies (goat anti-mouse FITC (A11001) and anti-rabbit AF555 (A21428), Invitrogen). Nuclei were counterstained with Hoescht 33342 (ThermoFisher Scientific). Images were acquired with a confocal microscope (Zeiss LSM 710) and analysed with ImageJ (Fiji).
Adipose tissue sections were stained for Mac2 (CL8942AP, Cedarlanelabs) overnight and then with the appropriate secondary antibody. Nuclei were counterstained with Hoescht 33342 (ThermoFisher Scientific). Slides were scanned using Zeiss Axio Scan Z1, and Mac2 staining was quantified with Visiopharm.

CD1 embryos were cultured until E4.5 from which 15 were selected with various sizes. To fluorescently label the DNA, the blastocysts were first incubated in KSOM-AA with 20 µM Hoescht 33342 (Thermo Fisher Scientific, Waltham, MA; 62249) at 37 °C for 30 min, and then KSOM-AA with 2 µM Hoescht 33342 at 37 °C for 30 min. The embryos were then imaged using the Operetta High Content Imaging System (Perkin Elmer, Waltham, MA) with the following parameters: 10 × objective; Hoescht excitation 50% and exposure 20 ms; bright field emission 10% and exposure 20 ms; focal plane in the center of the embryo. Using ImageJ 1.52p (NIH, USA), the images were cropped to 300 × 300 pixels. The background-subtracted Hoescht intensity was calculated as the average intensity of all pixels minus the median intensity of all pixels (plotted on the y-axis of Supplementary Fig. 6b). Each embryo was then analyzed by ddPCR and the cell count (calculations shown below) was plotted on the x-axis. Three empty wells were also imaged and analyzed as negative controls, all of which reported intensity and cell counts near 0.

To measure loss of membrane integrity, iCell hiPSC-CMs were co-stained for 15 min at 37°C with 0.3 μM DRAQ7 (Biostatus), a cell-impermeable dye, and with 0.8 μM Hoescht 33342 (Molecular Probes), as the membrane-permeable dye. Dyes were removed and the nuclear fluorescence scored (typically, 15,000-20,000 cells well^-1) using a Cellomics ArrayScan VTI High Content Screening platform. The line harbors a cardiomyocyte-specific Myh6-driven reporter gene for monomeric red fluorescent protein (RFP), enabling cell death to be scored exclusively in the myocyte population. Successfully transduced cells were identified on the basis of TurboGFP fluorescence. Images were analyzed with Developer XD, version 2.11 (Definiens).

Plants were grown according to the described; an upper leaflet was submerged within 10 ml of 1:2 diluted (from bulk) Fluorescein-containing liposomes for 72 hours, and leaflet samples were collected from adjacent leaves every 24 hours. Samples were thoroughly washed with 10% EtOH followed by DDW, and left within pre-prepared tissue-digestive enzyme solution over-night (O.N.) to facilitate protoplast isolation (modified protocol can be seen below). Prior to slide-mounting, additional fluorescent compound was added for nuclear staining (Hoescht 33342, λ_ex/λ_em = 350/453 nm, Molecular Probes^®, OR, USA) to a final concentration of 0.1 mg/ml.

ICC analyses were performed to evaluate the expression of genes related to pluripotency and differentiation. Before staining, all cell samples were preincubated for 10 min at 4°C and fixed with 4% paraformaldehyde for 30 min. After washing twice with Dulbecco’s phosphate-buffered saline (DPBS; Welgene), samples were treated for 1 h with 10% goat serum in DPBS to prevent nonspecific binding. Serum-treated cells were incubated overnight at 4°C with primary antibodies. The primary antibodies used were as follows: OCT4 (Santa Cruz Biotechnology, CA, USA; 1:200), SOX2 (Millipore; 1:200), NANOG (Santa Cruz Biotechnology; 1:200), SSEA1 (Millipore; 1:200), SSEA4 (Millipore; 1:200), Neurofilament (Millipore; 1:100), Vimentin (Millipore; 1:100), and Cytokeratin 17 (Millipore; 1:100). When we used the antibodies for intracellular proteins such as OCT4, SOX2, and NANOG, fixed cells were treated for 5 min with 0.2% Triton-X100 (Sigma-Aldrich, MO, USA) before serum blocking. After incubation with the primary antibody, the cells were treated for 3 h at room temperature with Alexa Fluor-conjugated secondary antibodies. Nuclei were stained with Hoescht 33342 (Molecular Probes). Images of stained cells were captured using a LSM 700 Laser Scanning Microscope (Carl Zeiss, Germany) and processed with the ZEN 2012 Light Edition program (Carl Zeiss).