Ki-67 Staining On Human PBMCs

September 26, 2017

BlueskyReddit
Anaesthesiology
LMU Munich
Researcher

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Quality of Results

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Company:

Biolegend

Product Name:

PE anti-human Ki-67 Antibody

Catalog Number:

350503

This antibody was used for detection of differences in the proliferation capacity of human PBMCs after treatment with certain inhibitors and cultivation for 5 days.

Experimental Design and Results Summary

Applications

Flow Cytometry

Sample

Human primary PBMCs

Primary Incubation

70% Ethanol @-20°C, add 3ml drop by drop while vortexing, incubate for 1h @ -20°C, wash 3x with FACS Buffer (PBS, 1%BSA)

Blocking Agent

None

Secondary Incubation

5µl antibody/million cells, incubation @ room temperature for 30 minutes in the dark, wash 2x with FACS Buffer

Tertiary Incubation

None

Detection

Flow Cytometry

Results Summary

Ki-67 antibody allows for staining of proliferating cells, since Ki-67 is expressed during cell proliferation phases such as M, G2 etc. Cells in G0 are not stained. Shift of the mean fluorescence intensity (MFI) may also be evaluated.

DOI or PMID #

N/A

Additional Notes

Cell size and granularity is affected by fixation and permeabilization with Ethanol. Perhaps gating adjustment compared to unfixed cells is needed.

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Summary

The Good

Only one antibody needed for detecting proliferation, rather simple, rather inexpensive, PE as a powerful conjugate

The Bad

Time-consuming fix/perm steps, excessive washing needed

The Bottom Line

For screening multiple conditions in an inexpensive way this antibody is really good. Staining is reproducible, antibody has high specificity, good value. Due to exzessive washing and long fix/perm steps this assay is not iseful for high throughput analysis.

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