Hoechst 33258 dye

Cyanidioschyzon merolae cells were grown and synchronized by 12/24 h light/dark regime. Cell fixation and permeabilization was performed, basically as described before (Hoff 2015 ). C. merolae cells were spun down and washed twice with 1x phosphate buffer saline (PBS) pH 7.2 (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 1.8 mM NaH₂PO₄). Cells were fixed with 4% formaldehyde for 10 min and washed 3× with 1× PBS buffer. Permeabilization was performed with 0.1% TritonX-100/PBS for 15 min and washed 3× with 1× PBS buffer. Blocking was performed with 5% BSA in 1× PBS for 45 min at RT. Next, cells were incubated at 4 °C overnight with primary antibody in blocking solution, then washed 4× with 1× PBS buffer. Afterwards, cells were incubated for 1 h at RT with secondary antibody in blocking solution and washed with 1× PBS buffer. Cells were next incubated for 1 h with 1 µg/mL Hoechst 33258 dye (Sigma-Aldrich, Germany) diluted in blocking solution and washed 6× with 1× PBS buffer. Cells were resuspended in 1× PBS buffer and placed on polilizyne microscope slides with equal volume of DAKO Fluorescent Mounting Medium (DAKO North America Inc., USA).
Primary and secondary antibodies were used at the following concentrations: 1:100 for rabbit anti-CAT antiserum, 1:100 for Alexa-488 goat anti-rabbit antibody (Thermo Fisher Scientific, USA).

Cell death was assessed by staining bEnd.3 cells with Hoechst 33258 dye (Sigma-Aldrich) and propidium iodide (PI; Sigma-Aldrich). Hoechst dye was added to the culture medium (10 μg/ml) and samples were then incubated at 37.5 °C for 30 min. PI solution was then added (5 μg/ml) just before cells were observed by confocal microscope (Carl Zeiss). PI-positive cells were counted as dead bEnd.3 cells.

DNA amount and sGAG content in the hydrogel disks were quantified after six weeks of implantation. The harvested cell-laden hydrogel disk implants were washed with PBS three times and freeze-dried. Each of the freeze-dried implants was digested by 500 µL papain solution (Sigma-Aldrich, St. Louis, MO, USA), which was prepared by dissolving papain at a concentration of 400 mg/mL in 0.1 M PBS (pH 6.0) containing 5 mM cysteine hydrochloride and 5 mM ethylenediaminetetraacetic acid (EDTA). 5 µL of the papain digestion solution was used to measure the DNA amount with Hoechst 33258 dye (Sigma-Aldrich, St. Louis, MO, USA). The fluorescence intensity was read with an FP-6500 spectrofluorometer (JASCO, Tokyo, Japan) at an excitation/emission wavelength of 360 and 460 nm. The sGAG content in each digestion solution was measured by using a BlyscanTM Glycosaminoglycan Assay Kit (Biocolor Ltd., County Antrim, UK). Four samples in each group were used for the measurement to calculate means and standard deviations.

To trace EVs by fluorescence microscopy, MSCs were labeled with Vybrant Cell Tracers Dil and Syto-RNA (Life Technologies, Carlsbad, CA) as previous described [20 (link)]. EVs obtained from labeled cells were concentrated and subjected to gradient separation as described above. For EV incorporation, mTEC were plated in 24-well plated and treated with different doses of labeled cCM-EVs (50,000, 150,000, 300,000 or 600,000 EVs/cell) for 24 h. Quantitative analysis of the EV uptake was conducted by FACS. To determine the incorporation of each fraction, mTEC were seeded into 24-well plates and incubated with labeled EVs (150,000 EVs/cell) from the different combined fractions for 24 h. The up-take of EVs was analyzed by microscope analysis using the ApoTome system (Carl Zeiss, Oberkochen, Germany). Hoechst 33,258 dye (Sigma-Aldrich) was added for nuclear staining.

Cells were seeded onto sterile glass coverslips in 24‐well dishes. Coverslips were washed once with PBS, fixed with 3.7% (w/v) formaldehyde, 200 mM HEPES pH 7.0 for 10 min and washed twice with PBS. Cells were permeabilized with 0.1% triton in PBS for 4 min. After two washes with PBS, samples were blocked by incubation for 30 min in blocking buffer (1% (w/v) BSA/PBS). Coverslips were incubated for 2 h at 37°C with primary antibodies in blocking buffer and washed three times in PBS. Coverslips were then incubated for 1 h at room temperature with Alexafluor coupled secondary antibodies (Life Technologies) in blocking buffer and washed an additional three times in PBS. If needed, nuclei were counterstained with 1 μg/ml Hoechst‐33258 dye (Sigma) for 5 min, and slides were washed twice with PBS and mounted in ProLong Gold (Invitrogen). Observations were made with Nikon Eclipse Ti widefield microscope (Plan Apo Lambda 60× Oil Ph3 DM) or a Zeiss LSM880 Airyscan Confocal Scanning microscope (ZEISS, 63× objective, NA 1.4). Images were processed using ImageJ and Adobe Photoshop Software.

Mice were transcardially perfused with a phosphate-buffered saline (PBS) and a 4% paraformaldehyde solution under anesthesia 6 weeks after the rAAV injection. The brains were removed and post-fixed with 4% paraformaldehyde for 6 h and immersed in 30% sucrose in PBS for 2 days. Sequential 14-µm slices were prepared on a cryostat (Thermo Scientific, Waltham, MA, USA). The cell nuclei were stained with a bis-benzimide solution (Hoechst 33258 dye, 5 µg/mL in PBS, Sigma-Aldrich, Darmstadt, Germany). Finally, the sections were mounted in an antiquenching medium (Fluoromount G; SouthernBiotech, Birmingham, AL, USA), followed by microscopy under a Zeiss AxioImager2 microscope with 10× and 40× air-immersion objectives.