Expression of CD1d on peripheral blood monocytes from patients with a rare autoimmune disease

Surgery
George Washington University, Washington DC
Post-doctoral Fellow

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Company:

BioLegend

Product Name:

PE/Cy7 anti-human CD1d Antibody

Catalog Number:

350310

CD1d is a lipid-antigen presenting molecule expressed by monocytes which binds and activates NKT cells. Antigen presenting cells may express CD1d as a molecule of stimulation of NKT cells and mediate inflammatory response. Here we measure the expression of CD1d as a measure of activated monocytes/ APC's and possible NKT cell stimulation. Peripheral blood was collected from patients with a rare autoimmune disease and expression of CD1d was analyzed on CD16+ monocytes/APC's. The high percentage of CD16+/CD1d+ cells reflected the population of activated monocytes /NK cells.

Experimental Design and Results Summary

Applications

Flow Cytometry

Sample

Human peripheral blood

Primary Incubation

100 ul human peripheral blood was added too a 50ul final volume of staining buffer containing anti-CD16 PercpCy7 and PECy7 anti-human CD1d antibody both in the ration of 1:100 (0.5ul in 50ul of staining buffer for each antibody.) Cells were incubated in dark for 30 mins and washed twice with staining buffer.Next RBC lysis was done by adding 2 ml of1X lysis buffer in each sample. Cells were kept in lysis buffer for not more than 15 mins. and washed again in staining buffer. Finally cells were re-suspended in 500ul of staining buffer for flow cytometric analysis.

Blocking Agent

NA

Secondary Incubation

NA

Tertiary Incubation

NA

Detection

Flow cytometric detection of PECY7 conjugated anti-CD1d human antibody

Results Summary

We found an increased percentage of CD16high/CD1d+ monocytes in patient samples compared to controls. clear distinct population was visible after analysis which meant that the disease group had an altered population of monocytes which represented activation and immune dysregulation.

DOI/PMID #

NA

Additional Notes

Antibody concentration and RBC lysis time needs standardization depending on the starting material

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Summary

The Good

Very bright staining antibodies

The Bad

Nothing to mention

The Bottom Line

Good method of analysis of distinct monocyte population or NKT cells using PECy7 anti-human CD1d antibody

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