Osteosomes and control liposomes were labeled with the red fluorescent lipophilic dye PKH26 (InVitrogen)23 (link). Next, PKH26-labeled osteosomes or liposomes (3×105 particles) were added to prostate cancer cells (1×104 cells), C4-2b or PC3-mm2, in RPMI1640 containing exosome-depleted 0.1% FBS, and cells were plated on a glass-bottom dish (ibidi). Exosome or liposome uptake into cells was observed by live-cell imaging on a BioStation (Nikon), in which images were captured every 30 min over 30 h using both bright-field and red fluorescence channels24 (link). For antibody blocking, PKH26-labeled osteosomes were preincubated with anti-Cad11 monoclonal antibody 1A525 (link) at a final antibody concentration of 3 μg/ml before the osteosome-antibody mixture was added to prostate cancer cells for live-cell imaging analysis. PBS buffer alone and an unrelated antibody with similar IgG isotype were used as negative controls.
Protocol detail
Tracking Osteosome Uptake by Prostate Cancer Cells
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Antibody
Buffer
Cell
Dish
Exosome
Fluorescence
Fluorescent dye
Igg antibody
Isotype
Liposome
Monoclonal antibody
Pkh26
Prostate cancer
Corresponding Organization :
Other organizations : The University of Texas Health Science Center at Houston, Center for Translational Molecular Medicine, The University of Texas MD Anderson Cancer Center, Baylor College of Medicine, Society of Surgical Oncology, Spanish Oncology Genitourinary Group
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Variable analysis
independent variables
- Treatment with PKH26-labeled osteosomes or liposomes
- Preincubation of PKH26-labeled osteosomes with anti-Cad11 monoclonal antibody 1A5
- Uptake of PKH26-labeled osteosomes or liposomes into prostate cancer cells (C4-2b or PC3-mm2)
- Observation of exosome or liposome uptake into cells by live-cell imaging
- Prostate cancer cell lines (C4-2b and PC3-mm2)
- RPMI1640 medium containing exosome-depleted 0.1% FBS
- Glass-bottom dish (ibidi) for cell plating
- Positive control: PKH26-labeled liposomes
- Negative controls: PBS buffer alone and an unrelated antibody with similar IgG isotype
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