Measuring Viability Of Thawed Cord Blood Derived CD34+ Cells

March 13, 2018

Biomedical Engineering
University of California, Davis
Graduate Student

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Company:

Biolegend

Product Name:

Zombie Yellow

Catalog Number:

423103

We are interested in studying granulopoiesis of hematopoietic stem cells. Human cord blood is a rich source of CD34+ hematopoietic stem cells that can be expanded and differentiated down the granulocyte lineage. After thawing CD34+ cells, measuring viability and frequency of CD34+ cells is important. We used Biolegend's Zombie Yellow as a viability marker for this experiment. We selected this product because it is compatible with paraformaldehyde fixation and it resides in the violet 3 channel, avoiding compensation issues with common fluorophores.

Experimental Design and Results Summary

Applications

Flow Cytometry

Sample

Human Cord Blood Derived CD34+ Cells

Primary Incubation

2 "test volumes" per 50µl cell suspension in culture medium (10% FBS in StemSpan SFEMII)

Blocking Agent

None

Secondary Incubation

None

Tertiary Incubation

None

Detection

Flow Cytometry

Results Summary

50µl of cell suspension (100,000 cells per ml) that was left over after seeding cells for culture was stained with zombie yellow and other antibodies for 30 minutes on ice. Cells were washed with 450µl PBS, and samples were run on an Attune NxT flow cytometer. Viability was determined to be 95%.

DOI or PMID #

N/A

Additional Notes

N/A

Image Gallery

Summary

The Good

Can be fixed. Can stain simultaneously with antibodies.

The Bad

Manufacturers protocol states staining should be done without serum. I increase the concentration of the dye and stain in serum and still see good separation between live and dead, but I use more product this way.

The Bottom Line

I like this product. It is nice having a viability dye in the violet 3 channel. It allows me to easily create more complex flow cytometry panels.

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