CD45RO for Labeling of Human PBMCs

February 26, 2016

Anaesthesiology
LMU Munich
Researcher

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Company:

BioLegend

Product Name:

Brilliant Violet 605™ anti-human CD45RO Antibody

Catalog Number:

304237

This antibody was included in a panel for identifying effector memory T cell subpopulations in human primary PBMCs.

Experimental Design and Results Summary

Applications

Flow Cytometry

Sample

Human primary PBMCs

Primary Incubation

Fc Block 10 mins room temperature

Blocking Agent

TruStain FcX blocking solution (BioLegend)

Secondary Incubation

2µl of antibody on 2 millions cells, 30 mins on ice in the dark (antibody), wash three times afterwards with 2ml of FACS buffer (HBSS with 1%BSA and 0,01% Sodium Azide)

Tertiary Incubation

NA

Detection

Flow Cytometry

Results Summary

This antibody works with the dilutions mentioned above, although it stained the "unstained" population a littel bit, compared to the isotype (see attached picture). Because of that it is even more necessary to include an isotype control antibody. Since the CD45RO positive cells are emitting a stronger fluorescence than the "unstained" cells, this antibody works, but this antibody property needs to be kept in mind. More extensive washing might be needed.

Additional Notes

Be sure to Fc-Block samples (10 Minutes, room temperature)m, wash three times after staining.

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Summary

The Good

Reliable staining with quite quick incubation time, BV as a more "exotic" dye interferes less with "traditional" colors such as FITC, therefore these might be used for different stainings in the same tube.

The Bad

BV605 conjugate rather expensive, same clone available with different conjugations

The Bottom Line

As usual, BioLegend provides an easy to use antibody that works with the concentrations suggested by BioLegend. The unspecific staining of the CD45RO-negative cells might be an antibody or a sample-induced issue. Maybe a different antibody does not show this. Maybe more extensive washing is needed. This antibody is rather expensive because of the BV conjugation.

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