Polystyrene round bottom tube

HEK293T cells were seeded overnight in 24-well plates at a density of 1.5 × 10⁵ cells per well before transfection with spike mutants for 24 h. Cells were detached and blocked in PBS containing 1% BSA in 5-mL polystyrene round-bottom tubes (Corning) on ice for 30 min. Cells were then stained with 1 µg/mL anti-S2 pAb or rabbit IgG isotype control (AC005, Abclonal) for 1 h. Excess antibodies were washed with PBS, and all cells were subsequently stained with goat anti-rabbit secondary antibodies conjugated with AlexaFluor488 (ThermoFisher) before being fixed in 1% paraformaldehyde (PFA) without membrane permeabilization. Flow cytometry was carried out on FACS Celesta (BD Biosciences), and data analysis was performed with FlowJo 10.4 (LCC).

Flow cytometry analysis was performed at the Immunophenotyping Platform of the Research Institute of the McGill University Health Centre (MUHC‐RI). Early apoptosis and necrosis were evaluated in cells under propranolol treatment (0, 50, 100, and 200 mmol/L) using Annexin V‐FITC kit (Cell Signaling Technology) following manufacturer's instructions. Unstained cells were used as a negative control. For cell cycle analysis, 8 × 10⁵ to 1.5 × 10⁶ cells were seeded per well in a six‐well plate. Plated cells were then starved during 12 hours in a SF RPMI 1640 media and afterward incubated with 0‐200 µmol/L propranolol. Following the 12‐hour treatment, single cell suspensions were prepared and fixed with ice‐cold 70% ethanol during 1 hour at 4°C. Subsequently, cells were washed with phosphate buffered saline and suspended in 1 µL/mL Ribonuclease A (Thermofisher) for 30 minutes at room temperature. Cells were then transferred into 5 mL polystyrene round‐bottom tubes (Corning), and stained with Propidium iodide (PI) solution (Cat P4170; Sigma) for 30 minutes at 37°C protected from light. Cell cycle was analyzed with BD FACSCanto II system and the PI solution (Sigma) was detected in the blue 488 nm wavelength, 585/42 nm filter.

To assess global translation, we used a puromycin-based assay (SUnSET)^{45 (link)}. Briefly, fibroblast-derived iPSCs were grown in 60 mm dishes with 3 ml of E8 media (Gibco/ThermoFisher) and grown for 4–5 d at 37 °C in 5% CO₂. The cells were incubated for 20 min with 1 μg/μl puromycin (Sigma-Aldrich) in preconditioned media, lifted with Accutase (StemCell Technologies), and transferred to a 96-well round-bottom plate (Corning). The cells were prepared for intracellular staining following the manufacturer’s protocol (eBioscience), and stained with AlexaFluor^® 647 anti-puromycin antibody (Millipore Sigma) or AlexaFluor^® 647 mouse IgG isotope control clone MOPC-21 (Biolegend) for 1 h at room temperature. For flow cytometry, cells were resuspended in 100 µl of flow cytometry staining buffer (1× PBS, 2% FBS, 0.1% sodium azide), and transferred to 5 ml polystyrene round-bottom tubes (Corning). Flow cytometry was performed with the assistance of the University of Michigan Flow Cytometry Core using a BD Aria3 cytometer (BD Biosciences). Compensations were determined using single color stained and unstained cells. Median fluorescence intensity was determined for both control and puromycin-stained samples, and expressed as fold change vs. control^{45 (link)}.

Complement activation on kidney cells was studied by flow cytometry using HK-2 cells (ATCC, CRL-2190), an immortalized human proximal tubular epithelial cell (PTEC) line reported to be a suitable alternative to freshly isolated PTCs (46 (link)). Cells were cultured in Keratinocyte Serum Free Medium supplemented with bovine pituitary extract, human recombinant epidermal growth factor (all from Gibco) and 0.5% Penicillin-Streptomycin (Sigma-Aldrich). On the day of the experiments, the cells were detached with TryPLE Express Enzyme, washed with pre-warmed Hank's balanced salt solution (both from Gibco) with calcium and magnesium, and transferred to polystyrene round bottom tubes (Corning, USA). Data was collected using a Gallios flow cytometer and analyzed by the Kaluza software 1.2 (both from Beckman Coulter, USA). Values are reported as median fluorescence intensities (MFI). To ensure analysis of single cells, a forward scatter area vs. height gate was defined upon a uniform gated population on the forward vs. side scatter (see Supplementary Figure 1). A total of 10,000 events were recorded per experimental condition.

T cell isolation was performed using EasySep™ Human CD3⁺ T cell Isolation Kit (Stem Cell Technologies, Vancouver, Canada) by negative isolation, according to manufacturer protocols. Cryopreserved CBMC were rapidly thawed in a 37 °C water bath and washed in Phosphate-buffered saline (PBS) supplemented with 2%FCS and 1 mM EDTA (separation buffer). Viability was assessed by Trypan blue dye exclusion assay and found to be approximately 90%. Cells were resuspended in 0.25 to 2 mL of separation buffer, in 5 mL (12 × 75 mm) polystyrene round-bottom tubes (Corning, Cat #352058), maintaining a cell concentration of 5 × 10⁷ cells/mL. EasySep™ Human CD3⁺ T Cell Isolation Cocktail was added at 50 μL/mL of sample and incubated for 5 min. EasySep™ Dextran Rapid Spheres™ (50 μL/mL) were added and samples were reconstituted to 2.5 mL separation buffer. The T cells were retrieved using an EasySep™ Magnet (STEMCELL Technologies, Cat #18000), with purity consistently greater than 97%.

Single cell from thymus, lymph nodes, spleen, liver, lung and brain suspensions were prepared and surface-stained as described previously^{9 (link),69 (link)–71} . Briefly, the thymus and lymph nodes were dissected from mice, and single-cell suspensions were prepared by gently teasing the tissue with forceps and then passing it through nylon filters. The liver and brain were dissected from the body, cut into pieces, minced in PBS buffer and thereafter filtered through a 40 μm cell strainer. The lungs were cut into pieces and incubated with shaking (200 rpm) at 37 °C for 1.5 h in RPMI medium (Gibco) containing 5% FBS (Gibco), 160 μg/ml collagenase IV (Sigma) and 0.2 μg/ml DNase I (Shanghai Sanjie). Leukocytes were isolated from the liver and brain cell suspensions or digested lungs by density fractionation using discontinuous 40–70% (vol/vol) Percoll (GE Healthcare) gradients. The cells were distributed in 5 mL polystyrene round-bottom tubes (Corning, Inc.) and stained for 40 min at 4 °C with the indicated antibodies. Cell fluorescence was observed using a two-laser FACSCalibur (BD Biosciences), a five-laser BD LSRFortessa (BD Biosciences) and a three-laser CytoFLEX (Beckman) flow cytometers. The data were analyzed with FlowJo software (TreeStar, Inc., Olten, Switzerland). Subsets of thymocytes were sorted using a FACSAria II flow cytometer (BD Biosciences).