Bright Flourophore, Easily Separates Populations

March 29, 2019

Immunology
Columbia University
Graduate Student

Overall

Quality of Results

Ease-of-Optimization

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Company:

Invitrogen

Product Name:

EOMES Monoclonal Antibody

Catalog Number:

48-4875-82

The goal of this project was to look at expression of different transcription factors in CD8 T cells in tumors to look at how the modulate exhaustion. This antibody was used as part of a phenotyping panel looking at markers of exhaustion and how it relates to transcription factors like Tbet and Eomes.

Experimental Design and Results Summary

Applications

Flow Cytometry

Sample

Murine Prostate Tumor

Primary Incubation

1:200 for 20 mins at RT

Blocking Agent

N/A

Secondary Incubation

Fix/Perm at RT for 40 mins

Tertiary Incubation

ICS at RT for 30 mins

Detection

Flow Cytometry

Results Summary

For this experiment, mouse tumors were first isolated and digested of the tumor matrix, followed by staining and flow analysis. For this experiment, Eomes in eFlour 450 (detected in BV421) was used in conjunction with 11 other colors. The Eomes stain was performed as an intracellular stain with a fixation/permeabilization kit designed for transcription factors purchased from ThermoFisher. At 1:200, we saw good resolution which was good enough for separating Eomes hi and low populations. When doing flow analysis, cells were gated on single live cells that were CD45+ CD3+ CD8+ CD4-. Between the spleen and the tumor, there is a lot more eomes expression in the tumor, which has been linked to expression of more exhaustion markers like PD1 in chronic infection models.

DOI or PMID #

N/A

Additional Notes

Use the correct fixation/permeabilization reagent.

Related Categories

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Summary

The Good

Clear staining, easy compensation

The Bad

Must use transcrition factor fix/perm

The Bottom Line

Very good antibody for eomes in mouse samples, can be used in conjunction with other dyes (with easy compensation due to emission/excitation spectrum) for multicolor flow. Can be used to stain whole tumors as well. Easy to optimize if you have a spleen and a sample from either a tumor or other cell population that has been chronically activated.

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