Hoechst 33342

Similar to our previous report^{35 (link)}, to examine the cell morphology under confocal microscopy after 24 h of incubation, cells on each surface were fixed in 4% paraformaldehyde for 20 min. A 1 μL stock solution of phalloidin-488 (AAT Bioquest) was diluted in 100 μL of PBS containing 1% bovine serum as the main working solution for staining. The surfaces were also washed with PBS for three times before staining the nuclei with 1 μL of Hoechst 33342 (AAT Bioquest) in 1 mL of PBS buffer for 10 min. Upon completion of staining and washing, a drop of Fluoromount (NOVUS) was carefully aliquoted onto the surface and subsequently covered with a coverslip and sealed with nail varnish. The stained cells with actin filament were observed under confocal microscopy (LEICA SP8) at 40x magnification to examine the cell morphology.

For in vivo phagocytosis experiment, CTX-induced TA injury was established as mentioned above while MSCs-ApoEVs were pre-stained by PKH26 (Sigma, USA) according to the manufacturer’s protocol. The PKH26 labeled MSCs-ApoEVs were injected into left TA muscles 3 days after TA injury models were established. The TA muscles were collected and fixed, dehydrated, and embedded in optimal cutting temperature compound (Leica, Germany) 24 h after PKH26 labeled MSCs-ApoEVs injection. For immunofluorescence staining, 10 μm sections were incubated with mouse anti-myosin antibody (R&D, MAB4470) at 1:250 dilution overnight at 4 °C and then incubated with Alexa Fluor 488 labeled secondary antibody (Yeasen, 33112ES60) at 1:500 dilution for 2 h at room temperature and finally stained with Hoechst 33342 (Invitrogen, H21492) for 10 min at room temperature, all operations are carried out in dark environments. The fluorescence images were captured by laser scanning confocal microscope (Nikon, Japan).
For in vitro phagocytosis experiment, PKH26 labeled MSCs-ApoEVs were added to C2C12 myoblasts and incubated for 3 h. After that, C2C12 myoblasts were fixed, permeated, and then stained by 488-conjugated phalloidin (AAT Bioquest, 23115) for 1 h and Hoechst 33342 for 5 min at room temperature. The fluorescence images were captured by laser scanning confocal microscope (Nikon, Japan).

Blood from healthy sheep was centrifuged at 1000g for 15 min to remove the plasma and buffy coat. Red blood cells were lysed (150 mM ammonium chloride, 10 mM Tris; pH 7.5). Ovine neutrophils, polymorphonuclear leukocytes (PMNs) were pelleted at 1000 g and washed. Cells were suspended in RPMI medium with 10% fetal bovine serum and examined under a microscope. Cells with a purity of more than 98% PMNs and more than 99% viability, determined by fluorescein diacetate and propidium iodide (FDA/PI) staining using fluorescence microscopy, were deemed acceptable for subsequent use. PMNs (10⁶) were incubated for 2.5 h with the Mha supernatant. Then, 100 mL of the cells was fixed and stained with May-Grunwald–Giemsa, and the rest of the suspension was tested for extracellular DNA quantification. Cells incubated in the presence of the leukotoxin were centrifuged, and the supernatants were removed to add fresh medium and isolate NETs. The mixture was centrifuged at 1000 g for 5 min. Neutrophil extracellular DNA was quantified using Hoechst 33342 (AAT Bioquest, USA) solution at 5 μg/mL. Fluorescence was measured using a plate reader [18 (link)].

Primary chondrocytes were seeded at 8 × 10⁴ cells per well and cultured for 24 h in 24-well plates. Chondrocytes were pretreated with nicotine in the absence or presence of the α7-nAChR‒selective antagonist, MLA, for 1 h and then treated with 5 μM MIA for 4 h. Cells were washed with PBS three times and fixed in 4% paraformaldehyde for 30 min at room temperature (22 ± 2 °C). After fixation, the cells were stained with Hoechst 33342 (10 μg/mL; AAT Bioquest, Sunnyvale, CA, USA) for 20 min and observed under a fluorescence microscope (Zeiss AX10; Carl Zeiss Microscopy GmbH, Jena, Germany). Cells displaying highly-condensed and brightly-stained nuclei were regarded as apoptotic cells. The apoptotic index was defined as the ratio of apoptotic cell number to total cell number.

(S)-(+)-camptothecin (#C9911), Triton X-100 (#X100), paraformaldehyde (#P6148), potassium phosphate monobasic (#P5655), sodium phosphate dibasic (#S5136), and dimethyl sulfoxide (DMSO, #D8418) were purchased from Sigma-Aldrich (Prague, Czech Republic); staurosporine (#3510A) was purchased from Santa Cruz Biotechnology (Heidelberg, Germany); Hoechst 33342 (#17530) was purchased from AAT Bioquest (Sunnyvale, CA, USA); and fetal bovine serum (#16000–036) was purchased from Thermo Fisher Scientific (Waltham, MA, USA). Hydrogen peroxide (30% solution, #23980–11000), potassium chloride (#60130G1000), sodium chloride (#71380G1000), isopropyl alcohol (#17510), nitric acid 65% (#18980) and sulfuric acid 96% (#20450) were obtained from Penta (Prague, Czech Republic).

Thioflavin T (Sigma-Aldrich) stock solution (10 mM in water) was mixed with 1X dPBS to obtain a final working concentration of 10 μM (1:1000). Hoechst 33342 (AAT Bioquest, Sunnyvale, CA, USA) stock solution (1 mg/mL) was mixed with 1X dPBS to obtain a final working concentration of 1 μg/mL (1:1000). After co-staining both ThT and Hoechst 33342 for 30 min, the attached cells were washed with 1X dPBS three times. The ThT signal was detected at the excitation wavelength of 450 nm and an emission wavelength of 490 nm on a microplate reader SpectraMax iD3 (Molecular Devices). The Hoechst 33342 signal was detected at the excitation wavelength of 360 nm and the emission wavelength of 460 nm on a microplate reader. The protein aggregative signal was calculated as the OD ratio of ThT/Hoechst 33342 as normalized ThT fluorescence intensity.