Clearly Labels Living Tumor Cells

January 03, 2017

Duke University
Immunology
Predoctoral

Overall

Quality of Results

Ease-of-Optimization

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Company:

eBioscience

Product Name:

Cell Proliferation Dye eFluor® 670

Catalog Number:

65-0840-90

The goal of this research was to label primary tumor cells in vitro for use in downstream phagocytosis assays identify to be assayed by flow cytometry.

Experimental Design and Results Summary

Application

Flow Cytometry

Starting Material

Primary mouse B cell lymphomas

Protocol Overview

Tumor cells in suspension were collected and then washed twice. Cells were resuspended at 20 million cells per mL in 500 mL warm PBS. While vortexing, 500 mL of either warm PBS alone or warm PBS containing 200 nM, 1 uM, 2 uM, or 10 uM Cell Proliferation Dye prepared according to manufacturer's instructions was added. Cells were incubated for 10 minutes at 37 degrees Celsius. Cells were then washed in cold media (5 mL) three times. After washing tumor cells labelled with Cell Proliferation Dye, cells were washed and resuspendd in PBS containing Fc block (diluted 1:250) and LIVE/DEAD (diluted 1:5,000), using 100 uL for one million cells). Cells were incubated on ice in the dark for 20 minutes.

Tips

This reagent will need to be optimized for the particular cell population and flow cytometer being used.

Results Summary

Tumor cells were clearly labelled with Cell Proliferation Dye, with increasing concentrations of Dye yielding increasingly bright cell populations (empty histogram, no Cell Proliferation Dye; increased shading indicating increasing concentrations of Cell Proliferation Dye, 100 nM, 500 nM, 1 uM, 5 uM).

DOI or PMID #

N/A

Additional Notes

N/A

Image Gallery

Summary

The Good

Clearly labels living tumor cells.

The Bad

It is difficult to optimize for your cell population and number, and if you do not have a tissue culture vortex, cells cannot be maintained in a sterile environment if following the manufacturer's protocol.

The Bottom Line

It works well if cells do not need to be sterile and there is time and reagent available for optimization.

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