Breast Cancer Exosomes Uptake Quantification

Fluorescently labeled, TAMRA-exosomes (10–50μlu derived from breast cancer cells (MCF-7; Rab27b; MDA-MB-231) were added to recipient cells and monitored using EPI fluorescence. Excessive exosomes were washed by culture media at various time intervals. To quantify the cellular uptake of exosomes and for each experiment more than 50 cells were imaged. All the settings of imaging and processing were kept constant and the relative fluorescent intensities were calculated.

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Harris D.A., Patel S.H., Gucek M., Hendrix A., Westbroek W, & Taraska J.W. (2015). Exosomes Released from Breast Cancer Carcinomas Stimulate Cell Movement. PLoS ONE, 10(3), e0117495.

Publication 2015

Breast cancer Cells Culture media Exosomes Fluorescence

Other organizations : National Heart Lung and Blood Institute, National Institutes of Health, Ghent University Hospital

Top 5 similar protocols

Variable analysis

independent variables

Exosomes derived from different breast cancer cell lines (MCF-7, Rab27b, MDA-MB-231)

dependent variables

Cellular uptake of exosomes
Relative fluorescent intensities

control variables

Imaging settings
Image processing

controls

No positive or negative controls were explicitly mentioned.

Annotations

Based on most similar protocols

Use fluorescently labeled exosomes (TAMRA-exosomes) derived from breast cancer cell lines (MCF-7, Rab27b, MDA-MB-231) to monitor uptake by recipient cells (Protocol 1).

Wash excessive exosomes with culture media at various time intervals to ensure accurate quantification of cellular uptake (Input protocol).

Image more than 50 cells per experiment and keep imaging and processing settings constant to calculate relative fluorescent intensities (Input protocol).

Use PKH26 red fluorescent dye to label exosomes and incubate with recipient cells for 12 hours (Protocols 1, 3, 4).

Centrifuge labeled exosomes at 100,000 × g for 1 hour to remove any remaining dye before incubating with cells (Protocol 2).

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