Dil dye (Beyotime, C1036) was added to 50 μL of exosome solution and then incubated at 37°C for 30 min. Dil-labeled exosome solution (1 μg/μL) was added to BM-derived MDSCs, followed by incubation at 37°C for 48 h with or without JPYZXZ. The culture medium was discarded, and fixed samples were blocked with blocking buffer (Beyotime, P0260) for 1 h, washed twice with PBS, and then incubated with anti-PD-L1 (Proteintech, 66248-1-Ig, 1:200) overnight. Next, the sections were incubated with fluorescent secondary antibodies and DAPI for 10 min. In addition, we analyzed tumor tissue and lung tissue sections from mice using the primary antibodies against CD11b (Abcam, ab13357, 1:100), anti-Gr-1 (BioLegend, 108448, 1:100), and anti-PD-L1 (Proteintech, 66248-1-Ig, 1:200). The sections were examined using fluorescence microscope and measured using ImageJ. Moreover, tumor tissues were fixed in 4% paraformaldehyde and embedded in paraffin, and then stained with H&E.
Dil dye
Manufactured by Beyotime
Sourced in China
Dil dye is a fluorescent membrane stain used for labeling and visualizing cell membranes. It can be used to stain a variety of cell types, including bacteria, yeast, and mammalian cells. The dye intercalates into the lipid bilayer of the cell membrane, allowing for easy identification and tracking of individual cells under a fluorescence microscope.
Automatically generated - may contain errors
Lab products found in correlation
P0260, by Beyotime (1 mentions)
Confocal laser scanning microscopy, by Leica (1 mentions)
Bx53 fluorescence microscope, by Olympus (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (1 mentions)
Paraformaldehyde (pfa), by Boster Bio (1 mentions)
Ab8226, by Abcam (1 mentions)
Ab6785, by Abcam (1 mentions)
Tcs sp5, by Leica (1 mentions)
5 protocols using dil dye
1
Exosome Uptake and PD-L1 Expression
2
Labeling sEVs and Tracking Uptake
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The sEVs were labelled with Dil dye (Beyotime, Shanghai, China) after 15 min incubation at 4 °C. The stained sEVs were co-incubated with H9c2 cells for 6 h at 37 °C. The H9c2 cells were then labelled with Calcein-AM dye (Beyotime, China) for 10 min at 37 °C and imaged by confocal laser scanning microscopy (Leica, Wetzlar, Germany).
3
Isolation and Labeling of Astrocyte Exosomes
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Astrocytes were cultured in serum-free medium and exposed to X-ray radiation. Cell supernatants were collected 0, 2, and 6 h after irradiation. Exosomes were isolated from the cell medium by ultracentrifugation (LP-100, Beckman Coulter). Briefly, the cell medium was centrifuged, then supernatants were collected and subjected to gradient centrifugation to remove cell debris. Large vesicles were removed using a 0.22 μm Millex GP syringe filter and exosomes were harvested by ultracentrifugation at 100,000 × g for 120 min twice and stored at -80 ºC. Exosomes were identified under a transmission electron microscope, re-suspended in PBS, and incubated with Dil dye (Beyotime) at 37ºC for 15 min in the dark. After labeling, the exosomes were washed and stored.
4
Tracking Extracellular Vesicle Uptake in Fibroblasts
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Normal fibroblasts were seeded into a 24-well plate 1 day prior to the experiment. Next, 40 μg EVs were added with Dil dye (Beyotime Biotechnology Co., Ltd, Shanghai, China) to attain a final concentration of 25 μM, and then reacted at room temperature for 30 min. The unbound dye was removed by ultracentrifugation. Next, the cells were rinsed with PBS thrice and fixed with 4% paraformaldehyde (Boster Biological Technology Co., Ltd, Wuhan, Hubei, China) for 30 min. Afterward, the cells were treated with 0.1% TritonX 100 (Chongqing Huierli Biotechnology Co., Ltd., Chongqing, China) for 30 min, fixed with 1% bovine serum albumin (BSA) for 30 min, and incubated with the primary antibody β-actin (ab8226, Abcam) at 4°C overnight. The following day, the cells were incubated with the FITC-labeled secondary antibody (ab6785, Abcam) for 1 h. Finally, the nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; Beyotime) for 30 min, and later the cells were photographed at × 400 using a BX53 fluorescence microscope (Olympus) equipped with a camera. The images were analyzed using ImageJ Pro Plus 6.0 software.
5
Protein and Organelle Colocalization
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Colocalization of proteins and organelles was observed with a laser confocal scanning microscope (TCS SP5; Leica, Wetzlar, Germany) and Dil dye (# C1036; Beyotime Biotechnology, Shanghai, China) a red fluorescent probe was used for the localization of cell membranes. Determination of protein was consistent with the above immunofluorescence assay.
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