Samples were spun onto poly-l-lysine slides using Stat Spin CytoFuge 2 (Beckman Coulter; Brea, CA USA) set at 800 rpm for 4 minutes. One drop of mounting medium containing 4′,6-diamidino-2-phenylindole (DAPI) stain to visualize nuclear structures (Vectashield, Vector Laboratories, Burlingame, CA, USA) was placed onto the glass slide before adding glass coverslip and sealing with nail polish. Slides were imaged using a fluorescence light microscope (BIOREVO BZ7000; Keyence; Osaka, Japan) and a Zeiss LSM 710 NLO confocal microscope.
Biorevo bz7000
Manufactured by Keyence
Sourced in Japan
The BIOREVO BZ7000 is a high-performance digital microscope system designed for a wide range of applications in life science research and clinical settings. It offers advanced imaging capabilities, including fluorescence and phase contrast, to facilitate detailed observation and analysis of biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 710 nlo confocal microscope, by Zeiss (1 mentions)
Statspin cytofuge 2, by Beckman Coulter (1 mentions)
Pkh26 red fluorescent cell linker kit, by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (1 mentions)
Pkh26, by Merck Group (1 mentions)
Pkh67, by Merck Group (1 mentions)
3 protocols using biorevo bz7000
1
Imaging Cells with DAPI Staining
2
Labeling and Visualizing Exosome Uptake
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USC-Exo were collected and labeled using a PKH26 red Fluorescent Cell Linker kit (Sigma Aldrich; Saint Louis, MO, USA) according to the manufacturer’s instructions. Then, the USC-Exo pellets were resuspended in 1 ml of Diluent C, after which 1 μl of PKH26 dye diluted in 250 μl of Diluent C was added and the USC-Exo were gently mixed for 4 min. Subsequently, an equal volume of 1% BSA was added to the reaction system to stop the reaction. Then, the PKH26-labeled exosomes were ultracentrifuged at 100,000×g for 70 min, washed with PBS, and ultracentrifuged again to remove excess dye. HUVECs were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The PKH26-labeled USC-Exo were incubated with cultured human umbilical vein endothelial cells (HUVECs) and stained with 4′,6-diamidino-2-phenylindole (DAPI; Vectashield, Vector Laboratories, Burlingame, CA, USA) to visualize nuclear structures and were further analyzed with a fluorescence microscope (BIOREVO BZ7000; Keyence; Osaka, Japan).
3
Fluorescent Labeling of Exosomes
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USC-Exos were labeled with green fluorescent dye PKH67 (Sigma Aldrich; Saint Louis, MO, USA) and CD133+ USC-Exos were labeled with red fluorescent dye PKH26 (Sigma Aldrich; Saint Louis, MO, USA) according to the manufacturer's instructions. Immediately after resuspending the exosomes in 0.5 mL of Diluent C reagent, mix with 0.5 mL of Diluent C diluted PKH67 dye or PKH26 dye. After incubation at room temperature for 4 min, 1 mL of 1% BSA was added to each reaction to stop the reaction. Subsequently, all samples were washed with PBS and centrifuged at 100,000×g for 2 h at 4 °C to remove excess dye. The PKH67-labeled USC-Exos and PKH26-labeled CD133+ USC-Exos were respectively incubated with the cultured BMSCs for 12 h. After nuclei were stained with DAPI, exosome uptake was observed using a fluorescence microscope (BIOREVO BZ7000; Keyence; Osaka, Japan).
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