Real-Time Assessment of the Antifungal Activity of Primary Human Immune Cells

Published: February 29, 2024

Abstract

Source: Brunel, S. F. et al., Live Imaging of Antifungal Activity by Human Primary Neutrophils and Monocytes in Response to A. fumigatus.J. Vis. Exp. (2017)

This video demonstrates live cell imaging for the analysis of antifungal activity in human neutrophils using fluorescent Aspergillus reporter conidia. Initially, both red and magenta fluorescence are detected within neutrophils, while red fluorescence decreases over time, indicating the degradation of conidia and confirming the antifungal activity of neutrophils.

Protocol

All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human welfare and have been reviewed by the local institutional review board. 1. A. fumigatus Culture and Conditions Prepare glucose minimal agar medium as described. Adjust media to pH 6.5 using 1 M sodium hydroxide (NaOH) and autoclave at 120 °C for 20 min. Allow…

Disclosures

The authors have nothing to disclose.

Materials

Glycerol Sigma G6279 http://www.sigmaaldrich.com
T75 flask Greiner Bio-One 658 175 http://www.greinerbioone.com/
PBS Gibco 20012-019 https://www.thermofisher.com/
Tween-80 Fisher Scientific 10592955 https://nl.fishersci.com/
40 µm filter Thermo Fisher Scientific 22363547 https://www.thermofisher.com/
15 ml Falcon tube Greiner Bio-One 188 261 http://www.greinerbioone.com/
50 ml Falcon tube Greiner Bio-One 227 261 http://www.greinerbioone.com/
MilliQ Millipore QGARD00R1 Nanopure water works similarly. http://www.merckmillipore.com/
Biotin Molecular Probes B-6352 https://www.thermofisher.com/uk/en/home/brands/molecular-probes.html
DMSO Sigma D5879 http://www.sigmaaldrich.com
Streptavidin-AF633 Molecular Probes S-21375 AF633 is the only tested fluorophore so far that remains visible in phagosomes. https://www.thermofisher.com/uk/en/home/brands/molecular-probes.html
EDTA blood tubes Greiner Bio-One 455036 Any blood tubes with an anti-coagulating agent should work. http://www.greinerbioone.com/en/start/
Ficoll-Paque Plus GE Healthcare 17-1440-03 http://www3.gehealthcare.com/
Trypan blue Thermo Fisher Scientific SV3008401 https://www.thermofisher.com/
HBSS Gibco 14170112 https://www.thermofisher.com/uk/en/home/brands/gibco.html
CD14 microbeads Myltenyi Biotec 130-050-201 Other monocyte isolation methods can be used as well, we prefer this method as it gives a consistent high purity of monocytes without being labor intensive. http://www.miltenyibiotec.com/en/
MS Columns Myltenyi Biotec 130-042-201 See comment at CD14 microbeads. http://www.miltenyibiotec.com/en/
RPMI + Glutamax Gibco 72400-021 https://www.thermofisher.com/uk/en/home/brands/gibco.html
CO2 independent medium Thermo Fisher Scientific 18045054 Necessary if there is not sufficient CO2 exchange during imaging. https://www.thermofisher.com/uk/en/home
µ-slide 8 well glass bottom Ibidi 80827 This is the imaging dish that we use, however any glass imaging dish of proper size should work. http://ibidi.com/
UltraVIEW VoX 3D Live Cell Imaging System Perkin Elmer L7267000 Includes volocity software for acquisition and analysis. http://www.perkinelmer.com/

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Cite This Article
Real-Time Assessment of the Antifungal Activity of Primary Human Immune Cells. J. Vis. Exp. (Pending Publication), e21991, doi: (2024).

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