Macsquant vyb

To develop the click chemistry-based C3b-labeling protocol an E. coli MG1655 strain expressing mCherry (pFCcGi was a gift from Sophie Helaine & David Holden^{72 (link)}) was used. Bacteria were cultured in Luria broth with 50 μg/mL ampicillin at 37 °C until stationary phase. A 1:100 subculture of E. coli was then cultured overnight in the presence of ampicillin 50 μg/mL and 2 nM KDO-N₃^{34 (link)} (Jena Biosciences). The incorporation of KDO-N₃ in the LPS was verified by an overnight click incubation with DBCO-Alexa Fluor 488 (Jena Biosciences; Germany) detected by flow cytometer (MACSQuant VYB, Miltenyi Biotec; Germany). Serial dilutions of C3b-DBCO were incubated with KDO-N₃-labelled bacteria at 4 °C for 24 h, to avoid bacterial growth and preserve C3b. C3b-DBCO labeling was detected with FITC-labelled F(ab’)2 Goat anti-Human-C3 by flow cytometry. A GFP-expressing E. coli MG1655 strain^{73 (link)} was labelled with C3b-DBCO as reported above and used for bulk experiments. Culture of this strain required 30 μg/mL gentamycin for plasmid maintenance.

Slc4a11^+/+ corneal endothelial cells were trypsinized and stained in triplicate with Pacific Blue Annexin V kit with PI (#640928, BioLegend, San Diego, CA, United States) or MitoSOX (#M36008, Thermo Fisher Scientific) following manufacturer’s instructions. Cells were collected in 2 ml micro centrifuge tubes following filtration using CellTrics Fliters (#04-004-2,327, Sysmex, Gorlitz, Germany). Flow cytometry analysis was conducted on MACSQuant VYB (Miltenyi Biotech, Germany). 10,000 cells were collected for each acquisition. Data were analyzed using FCS Express (De Novo software, Pasadena, CA, United States).

HeLa cells were transduced with lentivirus containing the pHR-SFFV-dCAS9-BFP-KRAB vector together with polybrene (5 μg/ml, Sigma). Twenty four hours after lentivirus transduction, the medium was replaced and the cells were incubated for another 48 hours. HeLa cells were then sorted for the BFP-expressing cells using the BD FACSAria III cell sorter (CRUK Flow Cytometry Core Facility). The expression of BFP fluorescent proteins was detected using MACSQuant VYB (Miltenyi Biotec) and the data were analysed using the FlowJo v7.1 software. BFP-sorted HeLa cells were used for single cell cloning in 96-well plate (clonal cells) or to create a stable non-clonal cell population.

HEK293 cells transfected with GFP or RFP were harvested and resuspended in PBS containing 1% FCS and 0.1% (w/v) NaN₃ (FACS‐PBS). To perform intracellular staining of GA₈₀‐flag, 1–2 × 10⁶ cells/staining were fixed with 4% PFA for 10 min at 37°C, washed once with PBS, permeabilized with FACS‐PBS containing 0.1% (w/v) saponin (FACS‐saponin), and incubated with 4% goat serum for 10 min at 4°C to block unspecific binding sites. Cells were then incubated with saturating amount of anti‐DYKDDDDK/flag antibody (1:250) or rabbit IgG (1:250) as control for 30 min at 4°C in the dark, followed by a single wash and incubation with saturating amount of secondary antibody (Alexa Fluor 647‐labeled anti‐rabbit IgG) for 30 min at 4°C. Cells were then washed two times with flow cytometry buffer and analyzed using MACSQuant VYB (Miltenyi). Data analysis was performed using FlowJo vX software (Treestar).
To perform fluorescence‐activated cell sorting of transmitted hydrophobic DPR proteins in a co‐culture assay, HEK293 cells were transfected with RFP, GFP, or DPR‐GFP for 24 h and mixed in the indicated combination for additional 24 h. Double‐positive cells were sorted using a FACSAria Fusion (BD Biosciences) cell sorter and plated on poly‐D‐lysine‐coated coverslips for imaging 17 h later.

Single-cell fluorescence of 24 h yeast subcultures was analyzed with flow cytometry using the MACSQuant VYB (Miltenyi). With the yeast culture diluted in 10-time with water in 96-well microtiter plate, GFP fluorescence from the biosensor was measured using the B1 channel (excitation 488 nm and emission 525/50 nm). Data was collected on 30,000 cells for each sample and processed using the FlowJo software.

The levels of TNF-α (#558299), IL-6 (#558301), IL-1β (#560232), IL-10 (#558300), IFN-γ (#558296), IL-2 (#558297), IL-12p70 (#558303), RANTES (#558345), MCP-1 (#558342), KC (#558340), and MIP-1α (#558449) in BALF were measured using a cytometric bead array kit (BD Biosciences). Microbeads with different intensities coated with capture antibodies specific for each cytokine and chemokine were added and mixed with 50 μL BALF for 1 h in the dark. Following incubation, the phycoerythrin (PE) detection reagent was added and incubated for 1 h. The mixtures were washed and analyzed using a flow cytometer (MACSQuant VYB; Miltenyi Biotec, Bergish Gladbach, Germany). The active TGF-β in the BALF was measured using a commercial ELISA kit (#88-8350-77, Thermo Fisher Scientific) according to the manufacturer’s protocol without sample acidification.