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.
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
2. A. fumigatus Labeling and Preparation
NOTE: When performing this assay for the first time, prepare the parental Af293-A. fumigatus strain. Take samples of both strains in microcentrifuge tubes and label only one of each strain as described below. This will allow one to have control conidia with no color and conidia with a single color, either dsRed or Alexa Fluor 633 (AF633), to test the setup and equipment.
3. Isolation of Human Neutrophils and Monocytes
4. Live Cell Video Microscopy
NOTE: The choice of the microscope will depend upon what is available locally, but the microscope setup will need to include an inverted stage, an environmental chamber heated to 37 °C and appropriate excitation/emission filters for dsRed (532/561 nm) and AF633 (633 nm). FLARE conidia do not work with the 488 nm laser in lieu of the 532/561 nm laser for dsRed. When performing this experiment for the first time, use the control conidia with no color and single color to test for background fluorescence and bleed-through between the dsRed and AF633 channels.
The authors have nothing to disclose.
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/ |