Differentiation of M1 Macrophages from Human PBMCs / Monocytes

February 17, 2016

LMU Munich
Anaesthesiology
Researcher

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Company:

Miltenyi Biotec

Product Name:

Human GM-CSF, Premium Grade

Catalog Number:

130-093-862

Here, our aim was to to differentiate M1 Macrophages from human PBMCs after extracting monocytes with the Pan Monocyte Isolation Kit II, human using different concentrations of GM-CSF.

Experimental Design and Results Summary

Application

Cell Culture

Starting Material

Human monocytes derived from PBMCs

Protocol Overview

We used 50ng/ml for 3d, Detection: microscopy

Tips

None

Results Summary

After extraction of monocytes from human PBMCs using the Pan Monocyte Isolation Kit II (see different review), monocytes were cultivated with RPMI supplemented with 20%FCS, 1% Glutamin and HEPES. Then, 50ng/ml GM-CSF was added to the cell culture. After 3 days, the supernatant was removed carefully, the cells were washed once with PBS and RPMI with 20% FCS, 1% Glutamin, HEPES and 50ng/ml GM-CSF was added to the cells. After incubation for another 3d, cell differentiation was evaluated with microscopy, and, afterwards, RT-PCR.

Additional Notes

You might want to use "difficult to cultivate" well plates for primary cells. This significantly increases final macrophage differentiation yield. This cytokine is supplied in different qualities: Research grade, which is less expensive and premium grade, which is a little more costly. In premium grade, Miltenyi guarantees the same biologival activity from lot to lot. In differentiation experiments, you might want to use premium grade so you do not have to asses the biologival activity for every new lot.

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Summary

The Good

Different sizes, comes lyophilized, so you can dilute with PBS to the desired stock concentrations. Reproducible amount of active cytokine.

The Bad

A little expensive, extensive aliquotting needed, you need to avoid repeated freeze/thaw cycles.

The Bottom Line

Good cytokine, reliable, guaranteed biological activity by manufacturer.

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