OK Spectral Flow Antibody

Chemical/Biomedical Engineering
University of Toronto
PhD Candidate

Overall

Quality of Results

Ease-of-Optimization

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Company:

Thermo Fisher Scientific (Invitrogen)

Product Name:

Podoplanin Monoclonal Antibody (eBio8.1.1 (8.1.1)), PerCP-eFluor™ 710, eBioscience

Catalog Number:

46-5381-82

Clone Number:

N/A

We are interested in studying the mechanisms responsible for ensuring T cell self tolerance that rely on thyme and extra-thymic Aire expressing cells.

Experimental Design and Results Summary

Applications

Flow Cytometry

Sample

Mouse thymus

Primary Incubation

Live dead: Zombie NIR LD 1:4000 in 100ul FACS for 20 min at RT

Blocking Agent

2.4G2

Secondary Incubation

Surface stains: Pdpn 1:50 in 100uL FACS for 30 min at 4C

Tertiary Incubation

PFA (15 min RT) + FoxP3 Fix/Perm kit (30 min 4C), then intracellular stain in 100uL perm buffer for 1hr at RT

Detection

Cytek Aurora spectral flow cytometry

Results Summary

This antibody gives decent staining to separate immature mTECs from mature/post-Aire mTECs after titration. However, manufacturer supplied protocol gives only weak detection of Pdpn+ cells by spectral flow, requiring a higher than usual concentration.

DOI or PMID #

N/A

Additional Notes

N/A

Image Gallery

Summary

The Good

Can give good separation of positive and negative populations.

The Bad

Requires optimization to work on mTECs.

The Bottom Line

Useful antibody for broadly separating mature and immature mTEC populations.

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