Mouse anti tra 1 60

Most of the reagents used in cell culture were obtained from Gibco (UK) unless specifically indicated. The primary and secondary antibodies were as follows: Rat anti-SSEA-3 (Invitrogen, USA), phycoerythrin (PE)-SSEA-4 (eBioscience, USA), mouse anti-TRA1-60 (Millipore, USA), PE-OCT-4 (BD Pharmingen, USA), rabbit anti-Nanog (Abcam, USA), fluorescein isothiocyanate (FITC)-goat anti-rabbit Ig (Abcam,), FITC-goat anti-mouse (Biorad, USA), FITC-conjugated sheep anti-mouse Ig (Sina biotech, Iran), and HRP-conjugated sheep anti-mouse Ig (Sina biotech). Extracellular vesicles-specific antibodies were from SBI system biosciences, CA, USA. Extracellular vesicle-depleted fetal bovine serum was purchased from Gibco. PKH-26 dye labeling kit was from Sigma (USA). Annexin V/PI apoptosis kit, ECL, BCA protein assay kit and calcein-acetoxymethyl (cAM) were purchased from ebioscience (USA), GE healthcare (USA) and BD Pharmingen, respectively.

The iPSC colonies were dissociated using TrypLE™ Select (Gibco Life Technologies) for 5 min, washed in DMEM:F12 and centrifuged at 300 g for 5 min before removing the supernatant and fixing with 3.7% PFA in PBS on ice for 10 min. The cells were washed again, followed by further centrifugation at 300 g for 5 min. After blocking and permeabilizing in 5% goat serum plus 0.3% Triton-x-100 in PBS for 30 min on ice, the cells were incubated with primary antibodies diluted in 5% goat serum/PBS for 30 min on ice. Following a further 2–3 washes, the cells were incubated with species-specific secondary antibodies conjugated to Alexa Fluor (Life Technologies) in the dark for 30 min on ice. For conjugated antibodies, only one antibody incubation step was required. The wash protocol was repeated and the cells were resuspended in 2% FBS/PBS and analysed by a BD Accuri™ C6 flow cytometer (BD Biosciences, Ann Arbor, MI, USA). The antibodies used included: mouse anti-OCT4 (STEMCELL Technologies), mouse anti-SSEA4 (STEMCELL Technologies), mouse anti-TRA-1-60 (Millipore), and mouse anti-TRA-1-81 (Millipore). Isotype controls included anti-IgG2b, anti-IgG3, and anti-IgM. All isotype control antibodies were obtained from Life Technologies.

Cells were fixed with 4% paraformaldehyde (PFA) or ethanol (OCT-4), blocked in 10% fetal calf serum-PBT, and immunostained using the following antibodies: mouse anti-OCT3/4 (Santa Cruz Biotechnology), mouse anti-TRA-1-60 (Millipore), mouse anti-NESTIN (Millipore), rabbit anti-alpha-fetoprotein (AFP, Dako), mouse anti-smooth muscle actin (SMA, R&D systems), mouse anti-ACTC1 (Abcam), rabbit anti-NFATc4 (Santa Cruz), rabbit anti-MYL2 (Proteintech), mouse anti-MYL7 (abcam). Cells were then immunostained with the appropriate conjugated secondary antibodies (Alexa Fluor 568 or 488, Molecular probes-Invitrogen). Nuclei were counter-stained with Hoechst-33342 (Sigma-Aldrich) or DAPI (Invitrogen). Specificity of the staining was verified by the absence of staining in negative controls consisting of the appropriate negative control immunoglobulin fraction (Dako).

For intracellular staining, iPS cell colonies were fixed and permeabilized using the Cytofix/Cytoperm Kit (BD Biosciences) according to the manufacturer’s recommendation. iPS colonies were overnight stained with directly conjugated mouse anti-OCT4 Alexa Fluor 488 (Millipore, 1:100) mouse anti-NANOG Alexa Fluor 488 (Millipore, 1:100) and unconjugated mouse anti-SOX-2 (Millipore 1:100). The next day, secondary antibody staining with Alexa Fluor 647 (Life Technologies, 1:500) was performed on iPS colonies previously stained with unconjugated SOX-2 antibody. Subsequently, all iPS colonies were also stained with DAPI (NucBlue Fixed Cell Stain, Life Technologies) prior to fluorescent microscopic analysis. Besides assessing the presence of typical intracellular pluripotency markers, iPS colony surface marker detection was carried out using primary unconjugated mouse anti-TRA-1-60 (Millipore, 1:100) and mouse anti-TRA-1-81 (Millipore, 1:200) followed by secondary antibody staining with Alexa Fluor 488 (Life Technologies, 1:500) and DAPI live cell staining (NucBlue Live Cell Stain, Life Technologies) (data not shown). Fluorescence was monitored with a Zeiss Observer Z1 microscope.

iPSC colonies were grown for 3 days on Matrigel™-coated coverslips suitable for immunofluorescence staining. The cells were fixed in 4% paraformaldehyde for 10 min at room temperature, rinsed twice with phosphate-buffered saline (PBS) and permeabilized in 0.5% Triton X-100 diluted in PBS for 10 min. Samples were then blocked with 2% FBS in PBS for 10 min at room temperature. The primary antibodies added were for typical pluripotency markers rabbit anti-Oct3/4 (1:100; Santa Cruz, CA, USA), goat anti-Nanog (1:20; R&D, Minneapolis, MN, USA), mouse anti-Sox2 (1:100; Millipore, Santa Cruz, CA, USA), mouse anti-TRA 1–60 (1:100; Millipore), mouse anti-TRA 1–81 (1:100; Millipore), mouse anti-SSEA4 (1:100; Hybridoma Bank, Iowa City, IA, USA) and kept at room temperature for 1 h. The secondary antibodies were as follows: donkey anti-rabbit Cye 3 (1:100; Chemicon) and donkey anti-mouse/goat Alexafluor 488 (1:100; Invitrogen, Carlsbad, CA, USA). Cells were also stained with DAPI (1:1000; Boehringer, Mannheim, Germany) for nuclei detection, diluted in blocking solution, and added in darkness at room temperature for 1 h. To stain the nucleus, 0.15% DAPI was added. The coverslips with the stained preparations were then mounted on glass. The staining was visualized using laser scanning confocal inverted microscope (Axio Observer.z1, Zeiss, Oberkochen, Germany).