Single Cell Analysis and Sorting of Aspergillus fumigatus by Flow Cytometry.

Experimental results in fungal biology research are usually obtained as average measurements across whole populations of cells, whilst ignoring what is happening at the single cell level. Microscopy has allowed us to study single-cell behavior, but it has low throughput and cannot be used to select individual cells for downstream experiments. Here we present a method that allows for the analysis and selection of single fungal cells in high throughput by flow cytometry and fluorescence activated cell sorting (FACS), respectively. This protocol can be adapted for every fungal species that produces cells of up to 70 microns in diameter. After initial setting of the flow cytometry gates, which takes a single day, accurate single cell analysis and sorting can be performed. This method yields a throughput of thousands of cells per second. Selected cells can be subjected to downstream experiments to study single-cell behavior.

www.bio-protocol.org/e3993 Bio-protocol 11(08): e3993. DOI: 10.21769/BioProtoc.3993 Please cite this article as: Howell and Bleichrodt, (2021). Single Cell Analysis and Sorting of Aspergillus fumigatus by Flow Cytometry. Bio-protocol 11(8): e3993. DOI: 10.21769/BioProtoc.3993. 11. To obtain germlings ( Figure 1C), inoculate 20 ml of MM with 5 × 10 6 spores/ml from fresh spore solution in a 25-cm 2 culture flask. 14. The next day, harvest isotropically grown spores and germlings from the standing cultures. 15. Vigorously mix the cultures by shaking them from side to side. can be labeled with Calcofluor white. The specific working concentration (25 nM-1 µM) must be determined experimentally and can be checked using a fluorescence microscope. Alternatively, organelles such as mitochondria can be fluorescently labeled to assess their activity. Another option is to use genetically encoded fluorophores (Bleichrodt and Read, 2019). As an example, we here describe fluorescent labeling of the cell wall using Calcofluor white, which binds to chitin.
2. Pipet 1 ml, from the harvested culture produced as described above (Step A7 or A14) into a 1.5ml Eppendorf tube.
3. Spin the sample down for 10 min at 6,000 × g at RT to pellet the cells. 4. Remove the supernatant with a pipet tip. 5. Add 1 ml Calcofluor white working solution (250 nM) to the pellet and resuspend by vortexing. 6. Incubate for 10 min at RT and spin down to pellet cells for 10 min at 6,000 × g at RT.
7. Remove the supernatant with a pipet tip.
8. Wash the cells by adding 1 ml ST and vortexing.
9. Spin the sample down for 10 min at 6,000 × g at RT to pellet the cells. 10. Run the beads at a flow rate that enables 2,000-3,000 events per second to be visualized on the software. 11. Set a sort gate within the software, which ensures that all beads will be apportioned to the sort stream.
12. Adjust the drop delay to ensure >95% of Accudrop beads are present in the sort stream. The drop delay is the time period (in microseconds) required for a particle of interest (e.g., a cell or a spore) to travel from the point of identification by the software to the end of the intact stream.
To ensure this timing is set correctly, we utilize 6-μm diameter Accudrop fluorescent beads and 2. Prior to sorting, align the sort stream to the capture plate by adjusting the deflection plate voltages and ensure the test sort droplets are central to each well. This procedure will ensure that the single droplets, which contain individual spores, are directed to the correct well along the entire 96-well plate. Each sorted particle is assigned its own unique X and Y coordinates on the plate, and this information is recorded within the data set for recall later during analysis.  11. Now sort 1 cell per well from the daughter gate using index sorting.
12. Find each cell in the well using microscopy and score which wells exclusively contain single cells of the desired type. 13. In the flow cytometry software, select all the wells that contained a single cell and plot this data.
14. Now plot the events in the initial gate in a new window and draw a daughter gate on this data, so that all of the events fall in the new gate. 15. Steps F10-F14 should be repeated for each cell type individually (e.g., dormant spores, isotropically grown spores and germlings; Figure 1  H. Cleaning procedure 1. Following each analysis or sorting procedure, it is important to establish and run an effective decontamination protocol. We tested various protocols for spore survival, and the most effective protocol was implemented in these sorting experiments.
a. Backflush the sample line with system sheath fluid to ensure that a minimal number of spores remain in the sample lines.
b. Using a 5-ml tube, run 4 ml of 70% ethanol through the sample lines for 5 min. 6. Backflush the nozzle with Milli-Q water and leave the nozzle submerged. 7. Switch off all the lasers and shut down the system. 8. Remove the waste tank. Do not empty the waste tank until the next day to ensure that no organisms can grow in the waste.

Data analysis
Exported flow cytometry data files can be opened and visualized in FlowJo and exported as csv files. For optional statistical analysis of cell subpopulations and heterogeneity thereof we refer the reader to Bleichrodt et al. (2020) (see Materials and Methods section and Text S1). The code for statistical analysis in R can be obtained from https://github.com/rbleichrodt/Conidial-heterogeneity and run in Rstudio.

Notes
1. In principle, this protocol can be used with any flow cytometer that allows index sorting.