Fitc conjugated secondary antibody

Flow cytometry was used to identify fractions containing EVs according to their tetraspanin content and performed as reported before[16 (link)]. Antibodies anti-CD9 (1:10, Clone VJ1/20), anti-CD63 (1:10, Clone TEA 3/18), or polyclonal isotype (1:5000, Abcam (ab37355), Cambridge, UK) were added to samples an incubated at 4°C for 30 min. After two washes, beads were incubated with FITC-conjugated secondary antibody (SouthernBiotech, Birmingham, AL) and analysed in a FacsVerse flow cytometer (BD Biosciences, San Jose, CA). Approximately 10,000 beads/sample were acquired and analysed using the Flow Jo software (Tree Star, Ashland, OR). In all samples, the top three tetraspanin-containing chromatographic fractions (those containing EVs) were pooled and used in experiments thereafter.

MEL cells were fixed with 3% PHA and permeabilized with ice-cold 70% ethanol. After the washing and blocking step (with 0.5% BSA/PBS), the cells were incubated with Alexa Fluor^®488-conjugated antibody against phospho-histone H2AX (Ser139; Cell Signaling Technologies, Danvers, MA, USA) or primary antiphospho-p38 antibody (Cell Signaling Technologies, Danvers, MA, USA) for 1 h. For phospho-p38 detection, FITC-conjugated secondary antibody (BD Biosciences, Franklin Lakes, NJ, USA) was used for another hour. The intensity of fluorescence was measured by FACS Calibur. To induce γ-H2AX, control MEL cells were irradiated with γ-rays (4 Gy, 30 min) before staining.

Reprogrammed hiPSCs (CMC-Fb-003) colonies were dissociated using Tryple Express (Life Technologies) and washed with PBS containing 10% FBS. Cell suspension was then stained with stage-specific embryonic antigens SSEA-4 (813-70, 1:100; Santa Cruz Biotechnology) or TRA-1-81 (TRA-1-81, 1:100; Santa Cruz Biotechnology) surface antibodies for 30 min. Intracellular staining for NANOG (1E6C4, 1:100; Santa Cruz Biotechnology) was performed by sequential incubations with fixation and permeabilization solutions (A and B Fix & Perm Solutions, Invitrogen, BD BioScience). These cells were incubated with FITC-conjugated secondary antibody (BD BioScience). Appropriate isotype controls were used for gating purposes (IgG3 isotype control to FITC (SSEA-4), IgM isotype control to FITC (TRA-1-81), IgG1 isotype control to FITC (NANOG)). Cells were analyzed using a FACS Canto II flow cytometer (BD Biosciences). Data were analyzed using a FlowJo software (TreeStar Inc., OR, USA).

We performed a flow cytometry analysis to compare the differentiation rate of BMMSCs to cardiomyocytes in different groups. The cells were first trypsinized, washed twice with PBS, and aliquoted into 1 × 10⁶ cells/100 μL in Fluorescence Activated Cell Sorting (FACS) tubes. 0.5 mL of cold flow cytometry fixation buffer (Shanghai Yeasen Biotechnology Co., Ltd, Shanghai, China) was added and the cells were incubated at room temperature for 10 min. The cells were washed twice with PBS and then resuspended in 1.5 mL of cold permeabilization wash buffer (Yeasen Biotech Co., Ltd, Shanghai, China) and vortexed. The cells were then incubated at room temperature for 10 min. The cells were centrifuged for 5 min at 1000 ×g. The supernatant was removed, discarded, and then incubated with cTnI antibody for 1 h. After washing twice, the cells were stained with a FITC-conjugated secondary antibody (BD Biosciences) for 30 min. For the negative controls, the cells were incubated only with a FITC-conjugated secondary antibody. Finally, cell suspensions were fixed in ice-cold 2% paraformaldehyde for flow cytometric analysis. Fluorescence data were collected using an EPICS XL Flow Cytometer (Beckman Coulter) and analyzed using the EXPO32 ADC software (Beckman Coulter).

Exosomes were isolated from PS Capture™ Exosome Flow Cytometry Kit (FUJIFILM Wako Chemicals) according to the manufacturer’s instructions. In brief, CSF or plasma were incubated with exosome binding enhancer and exosome capture beads at room temperature for 1 h. Exosomes were washed and resuspended in wash buffer for immunostaining. For RIP140 staining, exosome surface marker CD9 (Biolegend, clone: MZ3) was first labeled at room temperature for 1 h. Exosomes were later fixed and permeabilized with fixation/permeabilization solution kit (BD Biosciences, #554714). Briefly, exosomes were fixed in fixation/permeabilization solution at room temperature for 30 min and subsequently permeabilized with 1× permeabilization buffer and centrifuge at 400G for 10 min twice. After blocking with 100 μl 2% BSA, anti-mouse CD16/32 (BD Biosciences, clone: 2.4G2) in 1× permeabilization buffer at room temperature for 15 min, 100 μl RIP140 antibody (Abcam, ab42126) or isotype control (Abcam, ab37415) were directly added into the mixture and further incubated at room temperature for 1 h. Exosomes were later stained with FITC-conjugated secondary antibody (BD Biosciences, #554020) for 40 min on ice, then washed and resuspended in FACS buffer and analyzed by BD™ LSR II flow cytometry and analyzed by the FlowJo® software.