Flow Cytometry Staining of Human PBMC with Monoclonal Mouse Anti-Human NKp46

June 14, 2016

Division of BioMedical Sciences, Faculty of Medicine
Memorial University of Newfoundland
Research Assistant

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Company:

Santa Cruz

Product Name:

NKp46 Antibody (4k12): sc-71710

Catalog Number:

sc-71710

Nkp46 has been proposed as a universal marker for natural killer (NK) cells across species. The general aim of this experiment was to identify Nkp46+ cells from human PBMC. This product was chosen because it is recommended for detection of NKp46 of human origin by flow cytometry (as well as by western blot) by the manufacturer.

Experimental Design and Results Summary

Applications

Flow Cytometry

Sample

Human PBMC obtained from Ficoll density gradient separation

Primary Incubation

Anti-human Nkp46 antibody (Santa Cruz sc-71710), incubated with approximately 1x10^6 cells for 1 hour on ice at 1:10 dilution (1ug antibody) in a total volume of 100ul PBS+2% FCS, 1mM EDTA, 0.1% sodium azide.

Blocking Agent

N/A

Secondary Incubation

Alexa Fluor 647-conjugated donkey anti-mouse IgG (H+L) (Jackson 715 605 151), incubated for 1 hour on ice in the dark at 1:800 dilution in a total volume of 100ul PBS+2% FCS, 1mM EDTA, 0.1% sodium azide.

Tertiary Incubation

N/A

Detection

FACSCalibur (Becton Dickinson)

Results Summary

In the figure, cells stained with sc-717170 are represented by the black, open histogram, while isotype control cells are represented by the grey, filled histogram. The marker was set so that 1% of isotype control cells were positive for Nkp46.Approximately 10% of lymphocytes were identified as expressing Nkp46 at a weak to moderate level. Nkp46 staining was not as bright as desired, making it rather difficult to distinguish between Nkp46+ and Nkp46- cells without an isotype control performed in parallel. It should be noted that little optimization was performed here. Thus this product may perform better with optimization (antibody concentration, choice of secondary, etc).

Additional Notes

For flow cytometric analysis, lymphocytes were selected by gating on forward vs side scatter.

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Summary

The Good

Specific. Suitable for both western blot and flow cytometry (reported by the manufacturer)

The Bad

Requires a bit of optimization for optimal performance. Only available in one size and format (no fluorophore-conjugated versions available).

The Bottom Line

This product performs fairly well in identifying Nkp46+ cells from human PBMC by flow cytometry, but staining was not as bright as desired. Appropriate negative controls are essential when using this antibody in flow cytometry.

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