Cy3 conjugated affinipure goat anti mouse igg

The hucMSCs were seeded into a 6-well dish containing cover slips and incubated for 48 h. The cells were taken out and were then washed twice using PBS, fixed for 15 min with 95% ethanol, permeabilized in 0.1% Triton X-100 and blocked for 1 h using 1% BSA in PBST. Cells were then incubated with mouse anti-TRAIL antibodies (ProteinTech Group, Wuhan, China) at 4°C overnight. After that, slides were washed three times with PBS. Then cells were incubation with Cy3-conjugated affinipure goat anti-mouse IgG (ProteinTech Group, Wuhan, China) and DAPI (1 μg/ml; Thermo Fisher Scientific, Inc.) in the dark for 1 h at room temperature. After washing with TBS and distilled water, fluorescent images were collected using Olympus FV3000RS confocal microscope, and fluorescent signal was quantified using ImageJ.

Ginsenoside Rg1 (purity ≥ 98%, molecular structural formula is shown in Supplementary Figure S1) was obtained from Shanghai Tubei Biological Co., Ltd. The MTT kit was obtained from Sigma (United States). Annexin V-FITC/PI kit was obtained from BestBio (Shanghai, China). PI kit and RIPA lysis buffer were obtained from Beyotime Biotechnology (Shanghai, China). The BCIP/NBT alkaline phosphatase chromogenic kit and Alizarin Red S staining kit were obtained from Solarbio (Beijing, China). SYBR Green I Master Mix and reverse transcription kit were obtained from Thermo (United States). Anti-VEGF antibody was obtained from Boster (Wuhan, China). Anti-NOG, anti-Notch1, anti-CD31 and anti-Osterix antibodies were obtained from Abcam (United Kingdom). Anti-Emcn antibody was obtained from Abbkine (Wuhan, China). Horseradish enzyme-labeled goat anti-rabbit IgG and horseradish enzyme-labeled goat anti-mouse IgG were obtained from ZSGB-Bio (Beijing, China). Anti-GAPDH antibody, fluorescein (FITC)-conjugated AffiniPure goat anti-rabbit IgG and Cy3-conjugated AffiniPure goat anti-mouse IgG were obtained from Proteintech (United States). Rabbit anti-CD73/PE conjugated antibody, rabbit anti-CD90/PE conjugated antibody, rabbit anti-CD14/FITC conjugated antibody and rabbit anti-CD19/FITC conjugated antibody were obtained from Bioss (Beijing, China).

CFs cultured alone or in the presence of CMs were cultured on glass coverslips in 6-well plates. The cells were subjected to H/R as described above. Then, the cells were incubated with 4% fixative solution (Solarbio, China) for 30 min and blocked in 10% normal goat serum (Solarbio, China)/PBS for 30 min. The cells were incubated overnight at 4 °C with anti-NLRP3 (1:200), anti-ASC (1:200), anti-cleaved-caspase1 (1:200), anti-sarcomeric alpha actinin (1:200, Abcam, USA) and anti-Vimentin (1:200, Abcam, USA) primary antibodies. Thereafter, the cells were washed with PBS and incubated with goat anti-rabbit IgG/FITC (1:500, MultiSciences, China), goat anti-chicken IgG/Cy5 (1:500, Solarbio, China), and Cy3-conjugated Affinipure goat anti-mouse IgG (1:50, Proteintech, USA) secondary antibodies (30 min, room temperature) to visualize NLRP3, apoptosis-associated speck-like protein containing a CARD (ASC), cleaved caspase1, Vimentin and sarcomeric alpha actinin. We used 4′,6-diamidino-2-phenylindole (DAPI) (1:200, Beyotime, China) to visualize the nuclei. Finally, the fluorescence was visualized using a fluorescence microscope. Three fields of view were randomly selected to observe the fluorescent protein in each batch of cells by one blinded for allocation.