JAML-Staining Of Human PBMCs And Monocytes

September 20, 2017

Anaesthesiology
LMU Munich
Researcher

Overall

Quality of Results

Ease-of-Optimization

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Company:

R&D Systems

Product Name:

Human AMICA/JAML PE-conjugated Antibody

Catalog Number:

FAB34491P

I used this for adhesion studies of human PBMCs and Monocytes and evaluated JAM-L expression levels. To characterize the adhesive potential of different PBMC/Monocyte subpopulations and correlate it with JAM-L expression, this antibody was included in a FACS panel.

Experimental Design and Results Summary

Applications

Flow Cytometry

Sample

Human PBMCs and Monocytes

Primary Incubation

Fc Block 10 mins room temperature

Blocking Agent

TruStain FcX blocking solution (Biolegend)

Secondary Incubation

10µl/1million cells, vortex, Incubation for 30 Mins @ RT, dark, wash at least 3 times with FACS-Buffer (PBS 1%BSA)

Tertiary Incubation

None

Detection

Flow Cytometry

Results Summary

This antibody brightly stains human Monocytes for JAM-L. However, even after thorough washing the suggested Isotype exhibited some binding compared to unstained cells, although the Fc-Blockade was carried out. Therefore, be sure to sufficently Fc-block your sample. With this antibody, it is also possible to quantify for reduced expression levels of JAM-L after treatment.

DOI or PMID #

N/A

Additional Notes

Be sure to block with a blockig reagent due to unselective binding of the antibodies to monocytes caused by Fc Receptors. Therefore, always compare this antibody to the corresponding Isotype.

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Summary

The Good

Easy to handle, rather quick protocol, rather inexpensive antibody

The Bad

Excessive washing needed

The Bottom Line

Pretty good antibody for JAM-L staining on Monocytes. Reliable antibody, gives reproducible result. However, inour experiments the recommenden Isotype exhibited some light staining on human Monocytes (not PBMCs).

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