Controling For Background Signal In Flow Cytometry Utilizing APC-H7 Rat IgG2b, kappa Isotype Control

January 19, 2018

Pharmacology and Experimental Neuroscience
University of Nebraska Medical Center
Graduate Research Assistant

Overall

Quality of Results

Ease-of-Optimization

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Company:

BD Biosciences

Product Name:

APC-H7 Rat IgG2b, κ Isotype Control

Catalog Number:

560200

In this experiment T cells isolated from the spleen and negatively selected for CD4+ using R&D columns were incubated with cytokines and antibodies before being tested for phenotype using specific intracellular and extracellular antibodies and isotype controls.

Experimental Design and Results Summary

Applications

Flow Cytometry

Sample

CD4+ T cells from mice

Primary Incubation

0.1ug/1.0x105 cells for 30 minutes at 4 degrees celcius

Blocking Agent

20ug/ml gamma globulin

Secondary Incubation

N/A

Tertiary Incubation

N/A

Detection

BD LSRII Flow Cytometer

Results Summary

This antibody works moderately well at controling for background signal in respect to spcific antibody signaling, but it does have a tendency to give a high non-specific background signal that drowns out the antibody signal.

DOI or PMID #

N/A

Additional Notes

N/A

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Summary

The Good

This control does not require a lot of optimization

The Bad

This control shows high levels of background signal a good majority of the time.

The Bottom Line

This antibody can be used as a control, but its recommended a secondary control be used as well, such as an unstained control or a fluorescence-minus-one.

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