Hank s balanced saline solution

The assay was performed as previously described (57 (link)). 5 × 10⁵ HCC cells were plated at 6-well, and then cells were treated with rLECT2 in serum-free medium for 24 h. After cell harvesting, HCC cells were suspended in Hanks' Balanced Saline Solution (Gibco) containing with 5% fetal bovine serum (HyClone) and 5 μg/ml Hoechest 33342 (Sigma-Aldrich) for 90 min at 37 °C. In some cases, cells were incubated with Hoechst 33342 in the presence of 50 μM verapamil (Sigma-Aldrich) for reliable gating of SPCs. Subsequently, cells were centrifuged and resuspended in cold PBS containing 1 μg/ml propidium iodide (Sigma-Aldrich). SPCs were analyzed by flow cytometer (Beckman Coulter, Inc). The Hoechst 33342 was excited with the UV laser at 351 to 364 nm, and fluorescence was measured using a 515-nm SP filter (Hoechst blue) and a 608 EFLP optical filter (Hoechst red). A 540 DSP filter was used to separate the emission wavelengths.

Aspergillus fumigatus (strain AF4215, MYA 1163; American Type Culture Collection) was grown on Sabouraud glucose agar plates (BD Bioscience, San Jose, CA, USA) for three days at 37 °C. Conidia were collected in Hanks’ balanced saline solution (Gibco) and filtered through a cell strainer. The estimation of the conidial number was performed microscopically in a Neubauer slide (LO–Laboroptik, Friedrichsdorf, Germany). Resting conidia were immediately used or were stored at 4 °C for a maximum of one week. A. fumigatus hyphae were prepared by seeding of resting conidia in flat-bottom cell culture plates (Nunc, Langenselbold, Germany) in Yeast Nitrogen Base (Sigma-Aldrich, Taufkirchen, Germany) medium for 17 h at 37 °C.

Generation of single-cell suspensions for scRNA-seq was performed as follows: Skin biopsies were incubated overnight in 0.4% dispase (Life Technologies) in Hank’s Balanced Saline Solution (Gibco) at 4 °C. Epidermis and dermis were separated. Epidermis was digested in 0.25% Trypsin-EDTA (Gibco) with 10 U/mL DNase I (Thermo Scientific) for 1 h at 37 °C, quenched with FBS (Atlanta Biologicals), and strained through a 70 μM mesh. Dermis was minced, digested in 0.2% Collagenase II (Life Technologies) and 0.2% Collagenase V (Sigma) in plain medium for 1.5 h at 37 °C and strained through a 70 μM mesh. For the samples collected from the University of Pittsburgh, epidermal and dermal cells were combined in 1:1 ratio. For three of the samples collected from the University of Michigan, the epidermal and dermal cells were prepared in different libraries that were constructed by the University of Michigan Advanced Genomics Core on the 10X Chromium system with chemistry v3. For the remaining samples, the epidermis and dermis were combined in 1:1 ratio. Libraries were then sequenced on the Illumina NovaSeq 6000 sequencer to generate 150 bp paired-end reads. Data processing including quality control, read alignment (hg38), and gene quantification was conducted using the 10X Cell Ranger software. The samples were then merged into a single expression matrix using the cellranger aggr pipeline.

Generation of single-cell suspensions for scRNA-Seq was performed on 6 mm biopsies obtained from HS and healthy donors. Samples were incubated overnight in 0.4% dispase (Life Technologies) in Hanks balanced saline solution (Gibco) at 4°C. Epidermis and dermis were separated. Epidermis was digested in 0.25% trypsin/EDTA (Gibco) with 10 U/mL DNase I (Thermo Fisher Scientific) for 1 hour at 37°C, quenched with FBS (Atlanta Biologicals), and strained through a 70 μm mesh. Dermis was minced, digested in 0.2% collagenase II (Life Technologies) and 0.2% collagenase V (MilliporeSigma) in plain medium for 1.5 hours at 37°C, and subsequently strained through a 70 μm mesh. Epidermal and dermal cells were combined at a 1:1 ratio, and libraries were constructed by the University of Michigan Advanced Genomics Core on the 10X Chromium system with chemistry v2 and v3. Libraries were then sequenced on the Illumina NovaSeq 6000 sequencer to generate 150 bp paired-end reads. Data processing including quality control, read alignment (hg38), and gene quantification was conducted using the 10X Cell Ranger software. The samples were then merged into a single expression matrix using the cellranger aggr pipeline. See Supplemental Methods for information on cell clustering, cell type annotation, ligand-receptor analysis, pseudotime trajectory construction, and spatial transcriptomic analyses.

Two different strains each of A. fumigatus (strains AF4215, ATCC MYA 1163 and AF293), A. terreus (clinical isolates T9 and T90, provided by Cornelia Lass-Flörl, Medical University of Innsbruck, Innsbruck, Austria), A. niger complex (A. niger, clinical isolates BS and 715) and A. flavus (clinical isolates 253 and 467), and one strain of A. brasiliensis (strain ATCC 16404) were grown on Sabouraud glucose agar plates (BD Bioscience, San Jose, CA, USA) at 37 °C for 3 days. Conidia were harvested by gently scraping the surface of the plates, washed in Hanks’ balanced saline solution (Gibco, Paisley, UK) and filtered through sterile gauze. The number of the conidia was estimated in a Neubauer chamber (LO–Laboroptik, Friedrichsdorf, Germany). Resting conidia were used immediately for the experiments or stored at 4 °C for a maximum of one week. For the preparation of Aspergillus hyphae, resting conidia were plated in flat-bottom cell culture plates (Nunc, Langenselbold, Germany) and incubated in Yeast Nitrogen Base (YNB; Sigma-Aldrich, Taufkirchen, Germany) medium at 37 °C for 17 h to allow formation of hyphae.