M2-Mφ or mMSC-derived EVs were labeled with PKH26 (Sigma Aldrich, St. Louis, MO, USA) according to the manufacturer's protocols. mMφ cells were incubated with PKH26-labeled EVs (10 μg/mL) for 4 h at 37 °C with 5% CO2. After incubation, the cells were washed twice with PBS and stained with phalloidin (Yeasen Biotechnology, Shanghai, China) according to the manufacturers' instructions. The stained cells were observed using a confocal laser scanning microscope (N-STORM &A1, Nikon, Tokyo, Japan). To determine the cellular uptake mechanism of M2-EVs, cells were pretreated with inhibitors including Bafilomycin A1 (10 nM, Glpbio Technology, Montclair, CA, USA), Cytochalasin D (0.5 μM, Glpbio Technology), and Wortmannin (0.5 μM, Glpbio Technology) for 30 min, and then incubated with PKH26-labeled EVs. After incubation for 4 h, the cells were stained with phalloidin and observed using a confocal laser scanning microscope (Nikon).
Bafilomycin a1
Manufactured by GLPBIO
Sourced in United States
Bafilomycin A1 is a macrolide antibiotic that acts as a specific inhibitor of vacuolar-type H+-ATPase (V-ATPase). It is commonly used as a research tool to study cellular processes involving pH regulation and autophagy.
Automatically generated - may contain errors
Lab products found in correlation
3 protocols using bafilomycin a1
1
Uptake and Trafficking of M2-Derived EVs
2
Cellular Uptake Mechanisms of M2-EVs
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M2-Mφ or mMSCs-derived EVs were labeled with PKH26 (Sigma Aldrich, St.
Louis, MO, USA) according to the manufacturer's protocols. mMφ cells were incubated with PKH26 labeled EVs (10 μg/ml) for 4 h at 37°C with 5% CO2. After incubation, cells were washed twice with PBS and stained with phalloidin (Yeasen Biotechnology, Shanghai, China) according to the manufacturers' instructions. The stained cells were observed using a confocal laser scanning microscope (N-STORM &A1, Nikon, Tokyo, Japan). To determine the cellular uptake mechanism of M2-EVs, cells were pretreated with inhibitors including Bafilomycin A1 (10 nM, Glpbio Technology, Montclair, CA, USA), Cytochalasin D (0.5 μM, Glpbio Technology), and Wortmannin (0.5 μM, Glpbio Technology) for 30 min, and then incubated with PKH26labeled EVs. After incubation for 4 h, cells were stained with phalloidin and observed using a confocal laser scanning microscope (Nikon).
Louis, MO, USA) according to the manufacturer's protocols. mMφ cells were incubated with PKH26 labeled EVs (10 μg/ml) for 4 h at 37°C with 5% CO2. After incubation, cells were washed twice with PBS and stained with phalloidin (Yeasen Biotechnology, Shanghai, China) according to the manufacturers' instructions. The stained cells were observed using a confocal laser scanning microscope (N-STORM &A1, Nikon, Tokyo, Japan). To determine the cellular uptake mechanism of M2-EVs, cells were pretreated with inhibitors including Bafilomycin A1 (10 nM, Glpbio Technology, Montclair, CA, USA), Cytochalasin D (0.5 μM, Glpbio Technology), and Wortmannin (0.5 μM, Glpbio Technology) for 30 min, and then incubated with PKH26labeled EVs. After incubation for 4 h, cells were stained with phalloidin and observed using a confocal laser scanning microscope (Nikon).
3
Granulosa Cell Viability Assay
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Relative cellular viability was measured using the CCK-8 assay. Luteinized granulosa cells were seeded at a density of 5 × 104 cells in each well of 96-well plates and treated with different reagents as needed for 24 h. The reagent information is shown as follows: hydrogen peroxide (Hushi, Shanghai, China), resveratrol (Aladdin, Shanghai, China), rapamycin (Aladdin, Shanghai, China), 3-methyladenine (Aladdin, Shanghai, China), and bafilomycin A1 (GLPBIO., Shanghai, China). The cells were further incubated in 200 μL DMEM/F12 medium supplemented with 20 μL of CCK-8 reagent (Yiyuan Biotechnology, Guangzhou, China) for 2 h. The OD value was measured at a wavelength of 450 nm, and the relative cellular viability was normalized to the value of the control group.
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