Imaging CD14 positive human monocytes in peripheral blood mononuclear cells (PBMC) using wide-field microscope

Biochemistry
Weill Cornell Medical College
Research Associate

Overall

Quality of Results

Ease-of-Optimization

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Company:

Abcam

Product Name:

Anti-CD14 antibody [MEM-15] (FITC)

Catalog Number:

ab28061

Info: View Product View Product Specs

Browse Similar Products: CD14 Antibodies

We isolated peripheral blood mononuclear cells from fresh human blood using Ficoll Paque Plus protocol and stained them with anti CD14-FITC antibody (MEM-15) Abcam catalog # ab28061 to identify monocyte population. The fixed and anti CD14-stained cells were plated on Concanavalin A treated 96 well imaging plate. Images were acquired using Highthroughput ImageXpress wide field microscope.

Experimental Design and Results Summary

Applications

Immunofluorescence

Sample

Peripheral Blood Monocyte

Primary Incubation

45 min

Blocking Agent

2% Goat Serum in PBS for 15 min prior to FITC conjugated Primary antibody incubation

Secondary Incubation

NA

Tertiary Incubation

NA

Detection

Fluorescence Microscopy

Results Summary

Successfully able to image CD14 positive monocytes in mixed population of peripheral blood mononuclear cells isolated from fresh human blood using wide-field microscopy. Stained for nucleus using Draq5 to obtain per cell information Fixing and staining done in suspension. Plated after staining on Concanavalin A treated plate. Allowed to settle for 30 min and spin down the plate before imaging. I have tried using without block with any serum and found no non-specific staining compared to blocked cells. Using for microscopy, it makes it easy to monitor sub-cellular events in parallel by staining with second marker of interest.

Additional Notes

Normally, this is used for flow cytometery and FACS sorting

Related Categories

Image Gallery

Summary

The Good

Easy to use. Minimal non-specific staining.

The Bad

None

The Bottom Line

Best antibody.  Staining post-fix gives flexibility in terms of imaging 24-48 hrs after staining.  It's a one step process.

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