Isotype Control Antibody To Confirm Specific Antibody Binding

July 19, 2016

Pediatrics
Stanford University
Graduate student

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Company:

Biolegend

Product Name:

Alexa Fluor® 647 Rat IgG1, κ Isotype Ctrl Antibody

Catalog Number:

400418

The AF647 Rat IgG1 isotype control antibody was used as a control to ensure that the detection of intracellular IFN-gamma using an IFN-gamma antibody (clone XMG1.2) was not an artifact. The goal of the experiment was to measure the percentage of CD8+ T cells producing IFN-gamma after 3 days of stimulation by anti-CD3/CD28.

Experimental Design and Results Summary

Applications

Flow Cytometry

Sample

Mouse CD8+ T cells

Primary Incubation

N/A

Blocking Agent

N/A

Secondary Incubation

30 mins staining for surface receptors (CD8) on ice (1:200). Washed 3x with FACS buffer. Fixed and permed with 2% PFA and 4% sucrose for 30 min at RT before permeabilizing with 0.5% saponin.

Tertiary Incubation

Incubated Alexa Fluor® 647 Rat IgG1 antibody in perm buffer (0.5% saponin in FACS buffer) for 30 min on ice before washing 2x with perm buffer and 1x with FACS buffer.

Detection

Flow cytometry

Results Summary

Mouse CD8+ T cells were stimulated by anti-CD3/CD28 for 48 hours before being taken off and then re-stimulated with PMA/ionomycin at day 6. The CD8+ T cells were then fixed and permed and stained with either Alexa Fluor® 647 Rat IgG1 or anti-mouse IFN-gamma clone XMG1.2. Comparisons between the two stains showed that about 70% of the CD8+ T cells produced IFN-gamma.

Additional Notes

None

Image Gallery

Summary

The Good

Successfully serves as an isotype control for anti-mouse IFN-γ clone XMG1.2

The Bad

There was a small percentage of cells that expressed high levels of the isotype control antibody.

The Bottom Line

Overall, a great antibody to ensure specificity of whatever antibody used to detect intracellular cytokines.

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