Human CD3 eFluor 450 for FACS

August 22, 2017

Microbiology & Immunology
Columbia University
MD/PhD Student

Overall

Quality of Results

Ease-of-Optimization

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Company:

eBioscience™ from Invitrogen Antibodies

Product Name:

CD3 Monoclonal Antibody (OKT3), eFluor 450

Catalog Number:

48-0037-41

Human peripheral blood provides a useful source of various T cell subsets, whose functional status can be identified through a combination of surface receptor and transcription factor staining. We are interested in transcription factor expression by memory CD4+ and CD8+ human T cells, specifically amongst PBMCs. All of our multicolor panels involve staining for CD3 to identify T cells within the lymphocyte gate. The eFluor 450 conjugate was selected for multiplexing with larger panels to incorporate the violet laser.

Experimental Design and Results Summary

Applications

Flow Cytometry

Sample

Human PBMCs

Primary Incubation

1:200, 30min, 4C

Blocking Agent

FACS buffer (2% FBS in PBS, 2mM EDTA)

Secondary Incubation

N/A

Tertiary Incubation

N/A

Detection

BD Fortessa

Results Summary

PBMCs were obtained from freshly donated blood or thawed from frozen buffy coat specimens. Equivalent results were obtained with both sources of lymphocytes. Within the lymphocyte gate, a population of CD3+ cells was clearly identifiable when stained with the CD3-eFluor 450 antibody. Fluorescence intensity of the antibody and ease of separation was maintained following aldehyde fixation. Furthermore, this antibody was compatible with experiments that involved subsequent TCR (anti-CD3/CD28) stimulation following sorting.

DOI or PMID #

N/A

Additional Notes

N/A

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Summary

The Good

Strong separation; compatible with intracellular fixation/permeablization; reasonable dilution factor

The Bad

Not the cheapest available antibody

The Bottom Line

Solid antibody for multi-color panels that provides good, reliable separation and is compatible with subsequent TCR stimulations.

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