Cell proliferation was measured using the cck-8 kit (MCE, USA) and the EdU Cell Proliferation kit with an Alexa Fluor 596 Imaging kit (Thermo Fisher Scientific) following the manufacturer's instructions. In brief, normal hRECs or hRECs at an initial density of 5 × 103 cells/well, cocultured with 100 μg/mL MH-exo or PDR-exo, were seeded into 96-well plates and transfected with various oligonucleotides for 48 h. The cell proliferation was evaluated by 450 nm absorbance values at 6, 12, 24, 48, and 72 h thereafter using an enzyme-linked immunosorbent assay plate reader. Data are presented as mean ± SD of five replicates. Regarding the EdU assay, 100 μL 50 μM EdU medium was added into each well and incubated for 2 h in 37°C. The cells were then washed twice using PBS for 10 min, fixed in 4% PFA for 15 min, neutralized with 2 mg/mL glycine, and washed with PBS before permeabilizing with 0.5% Triton X-100 for 10 min. Finally, the hRECs were labeled using 100 μL Apollo-596 staining agent and washed in 0.5% Triton X-100 three times. EdU assay was performed three times independently and a total of three randomly selected fields were imaged in each condition using a confocal fluorescence microscope (MIC00223 LSM5 Live). The percentage of EdU-positive cells (labeled red) was calculated and analyzed using ImageJ software.
Alexa fluor 596 imaging kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Alexa Fluor 596 Imaging kit is a fluorescent labeling reagent designed for use in biological research applications. The kit provides a fluorescent dye that can be used to label and visualize target molecules or structures within cells or tissues. The core function of the kit is to enable fluorescent detection and imaging of the labeled targets.
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2 protocols using alexa fluor 596 imaging kit
1
Cell Proliferation Analysis of hRECs with Exosomes
2
Exosome-Mediated Regulation of hREC Proliferation
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Cell proliferation was assessed using the EdU Cell Proliferation Kit and the Alexa Fluor 596 Imaging Kit (Thermo Fisher Scientific), according to the manufacturer’s instructions. Briefly, hRECs in normal (5.5 mM glucose) or high-glucose medium (30 mM glucose) were seeded into 96-well plates at an initial density of 5 × 103 cells/well and were co-cultured with 100 mg/mL M0-exo or M2-exo for 24 h. Subsequently, 50 mM EdU medium was added to each well and incubated for 2 h at 37 °C. The cells were then washed twice with PBS for 10 min each. They were then fixed with 4% paraformaldehyde (PFA) for 15 min, neutralized with 2 mg/mL glycine, and then washed with PBS before permeabilization with 0.5% Triton X-100 for 10 min. Finally, the hRECs obtained were labeled with Apollo-596 stain; excess stain was removed by washing thrice with 0.5% Triton X-100. EdU assays were performed three times independently, and a total of three areas were randomly selected for confocal fluorescence microscopy imaging under both glucose conditions (MIC00223 LSM5 Live). The percentage of EdU-positive cells (red markers) was calculated and analyzed using the ImageJ software.
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