Great Antibody to Identify T-bet Positive Cell Populations

August 16, 2018

Oncology
Johns Hopkins School of Medicine
Graduate Student

Overall

Quality of Results

Ease-of-Optimization

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Company:

Biolegend

Product Name:

Brilliant Violet 421 anti-T-bet Antibody

Catalog Number:

644816

Our lab studies the role of mTOR signaling in CD8 T cell differentiation and function. T cell differentiation requires a dynamic change in transcriptional activity leading to programming. One key transcription factor, Tbet, is known to control effector T cell formation. Analyzing the level of Tbet is critical to determine any changes in this formation if there is a defect in effector function. We routinely stain for Tbet during the acute and memory stage to support out cytokine analysis. We used this particular antibody since it's been widely used and it readily identifies a nice distinct population.

Experimental Design and Results Summary

Applications

Flow Cytometry

Sample

Murine Splenocytes

Primary Incubation

1:250. 45 mins, Room temperature incubation (Intracellular staining using eBio Fix/Perm TF)

Blocking Agent

FC blocking in FACS buffer (2% FBS, .2% NaAzide/PBS) before fixing/perming

Secondary Incubation

None

Tertiary Incubation

None

Detection

Flow cytometer

Results Summary

We were able to determine a distinct Tbet+ CD8+ population in the heterogeneous spleen. Naive T cells did not express it; therefore, we knew this population was indeed real.

DOI or PMID #

N/A

Additional Notes

Make sure you use the Foxp3 transcription factor kit for staining since it is a transcription factor.

Related Categories

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Summary

The Good

The signal was very strong and was able to identify population using a nice dilution.

The Bad

Higher dilution needed to see the difference

The Bottom Line

Great antibody but definitely recommend titrating the antibody depending on the cell population and the state of the cell during the immune response. Typically get nice staining during the acute response of an infection.

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