Alexa 488

Flow-sorted chromosomes (10⁵) were cytospun (Cytospin3, Shandon) on to poly(l-lysine) slides at 1,200 r.p.m. for 10 min, as previously described^{36 (link)}. Samples were incubated with blocking buffer (5% normal goat serum in 10 mM Hepes, 2 mM MgCl₂, 100 mM KCl and 5 mM EGTA) for 30 min at RT, and incubated overnight at 4 °C with primary antibodies to Cenpa (catalog no. 2048S, Cell Signaling), H3K9me3 (catalog no. 07-523 or 07-442, Millipore), H3K27me3 (catalog no. ab6002, Abcam or catalog no. 07-449, Millipore), Esrrb (catalog no. PP-H6705-00, Perseus Proteomics) and Sox2 (catalog no. ab97959, Abcam). All antibodies were diluted 1:200 in blocking buffer. Chromosomes were incubated with appropriate secondary antibodies (anti-mouse Alexa 488 (catalog no. A11001, Invitrogen), anti-rabbit Alexa 488 (catalog no. A11008, Invitrogen) or anti-mouse Alexa 568 (catalog no. A11031, Invitrogen)) for 1 h at RT. All secondary antibodies were diluted 1:400 in blocking buffer. Stained chromosomes were mounted in DAPI-containing Vectashield (Vector Laboratories). Wide-field epifluorescence microscopy was performed on an Olympus IX70 inverted microscope using a UPlanApo ×100/1.35 oil objective lens and Micro-Manager software.

For single-labeling experiments with HRP, we used Alexa® 488 (goat anti-rabbit, Invitrogen A11034) or Cy3 (goat anti-rabbit, Dianova 111-165-003) each diluted 1:200. For double-labeling experiments, the combinations were α-ChAT (Cy3, donkey anti-rabbit, Dianova 711-167-003, dilution 1:200)/α-HRP (Alexa® 488, donkey anti-goat, Dianova 705-547-003, dilution 1:450) and α-vAChT (Alexa® 488, goat anti-guinea pig, Invitrogen A 11073, dilution 1:150)/α-HRP (Cy3, goat anti-rabbit, Dianova 111-165-003, dilution 1:150).

For quantification of γH2AX and H3S10p by fluorescence-activated cell sorting (FACS) cells were collected by trypsinization, fixed in 70% ethanol and permeabilized in 0.2% Triton X-100. Fixed cells were stained with primary antibodies diluted in PBS–1% FBS (mouse-anti-γ H2AX (1:500; Millipore, 05-636) or rabbit-anti-phospho-H3S10 antibody (1:500; Millipore, 06-570)) for 1 h followed by 1 h incubation with anti-mouse or anti-rabbit secondary antibody conjugated with Alexa488 (1:1,000; Invitrogen). For quantification of DNA-replicating cells by FACS, cells were pulse labelled with 40 μM BrdU before collection and ethanol fixation. For detection of total BrdU incorporation in double-strand DNA, fixed cells were treated with 2 M HCl (30 min) to denature DNA before a 2 h staining with mouse-anti-BrdU antibody (1:20; BD Biosciences, 347580) diluted in PBS–1% FBS followed by 1 h incubation with anti-mouse secondary antibody conjugated with Alexa488 (1:100; Invitrogen). DNA was stained using 0.1 mg ml⁻¹ propidium iodide supplemented with RNase A (20 μg ml⁻¹) for 30 min at 37 °C. Flow cytometry analysis was performed on FACSCalibur using CellQuest Pro software (BD). Quantification and analysis of cell cycle profiles were obtained using FlowJo (version 7.2.2; Tree Star, Inc.).

Goat anti-mouse immunoglobulins, conjugated to horseradish peroxidase (Sigma, A3673); used for western blotting in a 1:10,000 dilution. Chicken anti-mouse immunoglobulins conjugated to Alexa488 (Invitrogen; #A21200), used for IF microscopy in a 1:10,000 dilution. Donkey anti-rabbit immunoglobulins conjugated to Alexa568 (Invitrogen; #A10042), used for IF microscopy in a 1:10,000 dilution. Donkey anti-mouse immunoglobulins conjugated to Alexa568 (Invitrogen; #A10037), used for IF microscopy in a 1:10,000 dilution. Chicken anti-rabbit immunoglobulins conjugated to Alexa488 (Invitrogen; #A21441), used for IF microscopy in a 1:10,000 dilution. Monovalent Fab fragments rabbit anti-mouse (unconjugated; Fab rabbit anti-mouse IgG (H&L); Rockland Immunochemicals, #810-4102 via Biomol GmbH, Hamburg, Germany), used for IF microscopy in a 1:50 dilution.

All cells maintained in monolayer cultures were fixed with 4% paraformaldehyde for 10 min at room temperature. The pluripotency of the derived hiPSCs was tested by immunocytochemical staining for OCT4 (Abcam, Cambridge, United Kingdom; ab27985, RRID:AB_776898), NANOG (R and D Systems, AF1997, RRID:AB_355097), TRA-1–60 (Abcam, ab16288, RRID:AB_778563) and TRA-2–49 (Developmental Studies Hybridoma Bank, TRA-2-49/6E, RRID:AB_528071). The secondary antibodies were goat anti-rabbit IgG conjugated with Alexa-488 (Invitrogen, Carlsbad, California; A-11034, RRID:AB_2576217) and goat anti-mouse IgG conjugated with Alexa-488 (Invitrogen, A-11011, RRID:AB_2534069).

RUES2-CMs were first seeded on the ibidi µ-Slide 8-well chambered slides (Martinsried, Germany). The cells were fixed with 4% paraformaldehyde prior to being blocked with 5% bovine serum albumin (BSA). The cells were stained with α-actinin (1:200; Abcam, Cambridge, UK), cardiac troponin T (cTnT) (1:200; Abcam), and PD-L1 (1:100; Proteintech Group Inc., Chicago, IL, USA) antibodies and incubated overnight at 4 °C. The cells were incubated with appropriate secondary antibodies (Alexa-488 and Alexa-594; 1:500; Invitrogen). For CD4⁺ and CD8⁺ T-lymphocytes, cells were added to cytospin slide chambers with blotters and glass slides attached. The cells were then centrifuged on a glass slide using a cytocentrifuge. The staining process was done by adding the PD-1 antibody (1:100; Proteintech Group Inc., Rosemont, IL, USA) and then appropriate secondary antibodies (Alexa-488 and Alexa-594; 1:500; Invitrogen). 4’,6-diamidino-2-phenylindole (DAPI) was utilized to observe cells’ nuclei.