Gallios flow cytometer

Cell cycle analysis and apoptosis detection were carried out by flow cytometry [35 (link)]. Both HepG-2 and MCF-7 cells were seeded at 8 × 10⁴ and incubated at 37 °C, 5% CO₂ overnight, after treatment with the tested compounds, for 24 h. Cell pellets were collected and centrifuged (300 g, 5 min). For cell cycle analysis, cell pellets were fixed with 70% ethanol on ice for 15 min and collected again. The collected pellets were incubated with propidium iodide (PI) staining solution (50 µg/mL PI, 0.1 mg/mL RNaseA, 0.05% Triton X-100) at room temperature for 1 h and analyzed by Gallios flow cytometer (Beckman Coulter, Brea, CA, USA). Apoptosis detection was performed by FITC Annexin-V/PI commercial kit (Becton Dickenson, Franklin Lakes, NJ, USA) following the manufacture protocol. The samples were analyzed by fluorescence-activated cell sorting (FACS) with a Gallios flow cytometer (Beckman Coulter) within 1 h after staining. Data were analyzed using Kaluza v. 1.2 (Beckman Coulter).

Isolated neutrophils (10 × 10⁶ cells/ml in PBS without calcium and magnesium) were loaded with 10 μM carboxy-SNARF-1-AM (Thermo Fisher Scientific) and incubated at room temperature for 20 min followed by incubation at 37°C for 10 min. Cells were washed twice with PBS without calcium and magnesium and suspended in same buffer at 10 × 10⁶ cells/ml. Intracellular pH of neutrophils with different concentrations of bicarbonate in RPMI was recorded using Gallios Flow Cytometer (Beckman Coulter, USA). To determine the change in intracellular pH in response to extracellular pH of the medium, 100 μl of the cell suspension was added to 2.9 ml of respective medium preincubated at 37°C and 5% CO₂ and fluorescence was recorded for 1 min using Gallios Flow Cytometer (Beckman Coulter, USA) and respective extracellular pH was attained using predetermined volume of HCl and NaOH followed by flow cytometry measurement for 15 min. The change in intracellular pH was determined by ratio of FL6–FL2 in Beckman Coulter analysis software Kaluza 1.5.

For VSV-GFP experiments, cells were fixed with 4% Paraformaldehyde for 30 minutes at room temperature and analyzed for GFP fluorescence on a Gallios flow cytometer (Beckman-Coulter). For SARS-CoV-2 experiments, cells were fixed with 4% paraformaldehyde at room temperature for a minimum of 24 hours, washed once with PBS and permeabilized with 1X perm-wash buffer (BDBiosciences #554723) for 5 minutes. AlexaFluor 647-conjugated SARS-CoV nucleocapsid (N) antibody (clone 1C7C7, generously provided by the Center for Therapeutic Antibody Discovery at the Icahn School of Medicine at Mount Sinai) was diluted 1:400 in perm-wash buffer, and added directly to permeabilized samples, which were then incubated at room temperature for 40 minutes in the dark. Samples were washed once with 1X perm-wash buffer, once with calcium/magnesium-free PBS, and acquired on a Gallios flow cytometer (Beckman-Coulter). For all viral infections, analysis was performed with FlowJo software (Version 10.7.1, Becton Dickinson), excluding cell doublets and debris and gating according to mock infected populations (S6 Fig). Samples with fewer than 2000 cell events after doublet and debris gating were excluded from analysis.

T cell analysis was conducted using DuraClone IM T cell subsets tube (Cat. #B53328, Beckman Coulter). 1 x 10⁶ purified PBMCs were added to the tubes directly in 100µl and incubated at RT for 30 min in the dark. The samples were then pelleted at 300xg for 5min and washed once in 3 mL of PBS. The final samples were resuspended in 500 µl of PBS with 0.1% formaldehyde. Compensation for the assay was generated using the Compensation Kit provided in the IM DuraClone T cell subset tube using purified PBMCs.
B cell analysis was conducted using volunteer PBMCs using SARS-CoV-2 S-B Cell Analysis Kit, human (Miltenyi Biotec, 130-128-022). In short, PBMCs were stained with SARS-CoV-2 S-protein-Biotin was then then co-labelled with Streptavidin PE and Streptavidin PE-Vio 770 to eliminative the chance of non-specific binding. Cells were then stained with 7AAD, CD19, CD27, IgG, and IgM before analysed using FACS. All compensations were conducted using UltraComp eBeads™ Plus Compensation Beads (Cat. #01-3333-42, ThermoFisher). Samples were analysed using a Gallios flow cytometer (Beckman Coulter) and analysed using the Kaluza software (Beckman Coulter).
Samples were processed using a Gallios flow cytometer (Beckman) and the results were analyzed using the Kaluza Analysis software (ver 2.1, Beckman).