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

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…

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/