Anti-Ki-67 Set That Needs Optimizing

August 17, 2018

BlueskyReddit
Developmental Immunology
La Jolla Institute for Allergy and Immunology
Postdoctoral Fellow

Overall

Quality of Results

Ease-of-Optimization

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Company:

BD Biosciences

Product Name:

PE Mouse Anti-Ki-67 Set

Catalog Number:

556027

Our goal was to stain various T cell subsets for Ki-67 before and after infection, and complete this staining with transcription factors and BrdU labeling. We selected this antibody because it is a bright fluorophore and others had reported success with this antibody alone.

Experimental Design and Results Summary

Applications

Flow Cytometry

Sample

Collagenase-digested mouse lung tissue, including T cells

Primary Incubation

Cocktail of surface antibodies for 20 minutes on ice

Blocking Agent

Blocked with 24G2 Fc block in 3% BSA in PBS prior to surface staining

Secondary Incubation

1x Fix/Perm for 1 hour

Tertiary Incubation

1:200 in 1x perm buffer overnight at 4 degrees

Detection

Flow cytometer

Results Summary

The Ki-67 appeared as a gradient for all T cell subsets we looked at, without a clear peak. The use of the isotype control was particularly helpful to identify positive and negative populations. While we had to optimize the fix/perm protocol for Ki-67, this resulted in a reduction in transcription factor co-staining. Therefore it may require further optimization to find a strategy that works well for all intracellular markers.

DOI or PMID #

N/A

Additional Notes

N/A

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Summary

The Good

Can separate Ki-67+ and Ki-67- cells, particularly with the comparison with an isotype control

The Bad

Difficult to optimize with other intracellular markers

The Bottom Line

The antibody works well when optimized for this intracellular staining. If you are working with other difficult intracellular markers, such as transcription factors, the protocols will have to be re-optimized for co-staining.

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