ExpiCHO-S cells were seeded at 6 × 106 cells per ml in a volume of 5 ml in a 50-ml bioreactor. The following day, cells were transfected with SARS-CoV-2 S glycoprotein-encoding pcDNA3.1(+) plasmids (BetaCoV/Wuhan-Hu-1/2019, accession number MN908947, Wu-D614; Omicron BA.2, BQ.1.1, XBB.1, XBB.1.5, BN.1 or BA.2-E340A generated by overlap PCR mutagenesis of the Wu-D614 plasmid) harbouring the Δ19 C-terminal truncation. S-encoding plasmids were diluted in cold OptiPRO SFM (Life Technologies, 12309-050), mixed with ExpiFectamine CHO Reagent (Life Technologies, A29130) and added to cells. Transfected cells were then incubated at 37 °C with 8% CO2 with an orbital shaking speed of 250 rpm (orbital diameter of 25 mm) for 24 to 48 h. Transiently transfected ExpiCHO-S cells were harvested and washed twice in wash buffer (PBS 2% FBS, 2mM EDTA). Cells were counted and distributed into round bottom 96-well plates (Corning, 3799) and incubated with serial dilutions of mAb starting at 10 μg ml−1. Alexa Fluor 647-labelled Goat anti-human IgG secondary antibody (Jackson ImmunoResearch, 109-606-098) was prepared at 2 μg ml−1 and added onto cells after two washing steps. Cells were then washed twice and resuspended in wash buffer for data acquisition at Ze5 cytometer (Bio-Rad).
Ze5 cytometer
Manufactured by Bio-Rad
The ZE5 cytometer is a flow cytometry instrument designed for high-throughput analysis of cells. It features a compact design and advanced optics for sensitive detection and quantification of various cellular parameters.
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Lab products found in correlation
96 well round bottom plate, by Corning (11 mentions)
Expifectamine cho reagent, by Thermo Fisher Scientific (6 mentions)
Optipro sfm, by Thermo Fisher Scientific (4 mentions)
Alexa fluor647 labeled goat anti human igg secondary ab, by Jackson ImmunoResearch (4 mentions)
Tpck trypsin, by Worthington (3 mentions)
Alexa fluor647 labelled goat anti human igg secondary ab, by Jackson ImmunoResearch (3 mentions)
Prism v9, by GraphPad (2 mentions)
Zombie aqua fixable viability kit, by BioLegend (2 mentions)
Mini bioreactor centrifuge tube, by Corning (2 mentions)
Expicho expression medium, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 647 labelled goat anti human igg secondary antibody, by Jackson ImmunoResearch (1 mentions)
7 aad, by BioLegend (1 mentions)
Fcs express, by De Novo Software (1 mentions)
Facsmelody, by BD (1 mentions)
Lsrii flow cytometer, by BD (1 mentions)
Aldefluor kit, by STEMCELL (1 mentions)
Propidium iodide, by Thermo Fisher Scientific (1 mentions)
Rnase a, by Thermo Fisher Scientific (1 mentions)
Modfit lt software, by Verity Software House (1 mentions)
Rnase, by Thermo Fisher Scientific (1 mentions)
17 protocols using ze5 cytometer
1
SARS-CoV-2 Spike Protein Expression and Antibody Binding Assay
2
Kinetics of SARS-CoV-2 Spike Antibody Binding
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CHO cells stably expressing the prototypic SARS-CoV-2 S were harvested, washed in wash buffer (PBS 1% BSA 2 mM EDTA) and resuspended in PBS. Cells were then counted and 90’000 cells/well were dispensed into a round-bottom 96 well plate (Corning) to be treated with 10 ug/ml TPCK-Trypsin (Worthington Biochem) for 30 min at 37°C. After a washing step, cells were incubated with 15 µg/ml mAbs solution for 180, 120, 60, 30 or 5 min at 37°C. After the incubation for the allotted time, cells were washed with ice-cold wash buffer and stained with 1.5 µg/ml Alexa Fluor647-labeled Goat Anti-Human IgG secondary Ab (Jackson Immunoresearch) for 30 min on ice in the dark. Cells were then washed twice with cold wash buffer and analyzed using a ZE5 cytometer (Biorad) with acquisition chamber T= 4°C. Binding at each time point (MFI) was determined normalizing to the MFI at 5 minutes time point and data plotted using GraphPad Prism v. 9.1.1
3
Investigating SARS-CoV-2 Spike Glycoprotein Variants
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ExpiCHO-S cells were seeded at 6 × 106 cells/mL in a volume of 5 mL in a 50 mL bioreactor. The following day, cells were transfected with SARS-CoV-2 spike glycoprotein-encoding pcDNA3.1(+) plasmids (BetaCoV/Wuhan-Hu-1/2019, accession number MN908947, Wuhan D614; Omicron BA.1 and BA.2 generated by overlap PCR mutagenesis of the Wuhan D614 plasmid) harboring the Δ19 C-terminal truncation27 (link). Spike encoding plasmids were diluted in cold OptiPRO SFM (Life Technologies, 12309-050), mixed with ExpiFectamine CHO Reagent (Life Technologies, A29130) and added to cells. Transfected cells were then incubated at 37˚C with 8% CO2 with an orbital shaking speed of 250 RPM (orbital diameter of 25 mm) for 24 to 48 h. Transiently transfected ExpiCHO-S cells were harvested and washed twice in wash buffer (PBS 2% FBS, 2 mM EDTA). Cells were counted and distributed into round bottom 96-well plates (Corning, 3799) and incubated with serial dilutions of mAb starting at 10 μg/mL. Alexa Fluor647-labelled Goat Anti-human IgG secondary Ab (Jackson ImmunoResearch, 109–606–098) was prepared at 2 μg/mL and added onto cells after two washing steps. Cells were then washed twice and resuspended in wash buffer for data acquisition at ZE5 cytometer (BioRad).
4
NTD Antibody Binding Assay
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Transiently transfected ExpiCHO cells were harvested and washed two times in wash buffer (PBS 1% BSA, 2 mM EDTA). Cells were counted, distributed into round bottom 96-well plates (Corning) and incubated with the NTD antibodies at the final concentration of 5 μg/mL. Alexa Fluor647-labeled Goat Anti-Human IgG secondary Ab (Jackson Immunoresearch) was prepared at 1.5 μg/mL added onto cells after two washing steps. Cells were then washed twice and resuspended in wash buffer for data acquisition at ZE5 cytometer (Biorad).
5
Kinetic Analysis of SARS-CoV-2 Spike Binding
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CHO cells stably expressing the prototypic SARS-CoV-2 Spike protein were harvested, washed in wash buffer (PBS 1% BSA 2 mM EDTA) and resuspended in PBS. Cells were then counted and 90’000 cells/well were dispensed into a round-bottom 96 well plate (Corning) to be treated with 10 ug/ml TPCK-Trypsin (Worthington Biochem) for 30 min at 37°C. After a washing step, cells were incubated with 15 ug/ml mAbs solution for 180, 120, 60, 30 or 5 min at 37°C. After the incubation for the allotted time, cells were washed with ice-cold wash buffer and stained with 1.5 ug/ml Alexa Fluor647-labeled Goat Anti-Human IgG secondary Ab (Jackson Immunoresearch) for 30 min on ice in the dark. Cells were then washed twice with cold wash buffer and analyzed using a ZE5 cytometer (Biorad) with acquisition chamber T= 4°C. Binding at each time point (MFI) was determined normalizing to the MFI at 5 minutes time point and data plotted using GraphPad Prism v. 9.1.1
6
Thawing and Staining of PBMCs
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PBMCs were thawed and washed twice with RPMI-1640 W/O L-Glutamine (10×500ml) (Life Technologies Europe BV, 31870074) 10% HyClone Fetal Bovine Serum, US Origin 500ml (VWR International, SH30070.03), and incubated in the same medium for 2 h at 37°C. Live PBMCs were counted post thawing and seeded at 1 million into round-bottom 96-well plates (Corning, 3799). PBMCs were stained with LIFE/DEAD marker (Zombie Aqua Fixable Viability Kit, BioLegend 423101) in Dulbecco’s phosphate-buffered saline (DPBS) w/o Ca and Mg (500ml), (Chemie Brunschwig, P04–36500 Pan Biotech) for 30’ at RT, washed in MACS buffer (PBS 2% HyClone, 2 mM EDTA), and stained with antibodies to CD3, CD4, CD19, CD16, CD14, (BioLegend), IgG (BD Bioscience) (Key Resources Table ) for 30’ at 4°C. Cells were then washed and resuspended in MACS buffer for data acquisition at ZE5 cytometer (Bio-Rad). Data were analysed with FlowJo software.
7
ALDH Activity Assay Protocol
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ALDH activity was examined using the ALDEFLUOR™ kit (Stem Cell Technologies) according to the manufacturer’s protocol. Briefly, single-cell suspensions were incubated with either the ALDEFLUOR™ reagent alone or the ALDEFLUOR™ reagent and the ALDH inhibitor diethylaminobenzaldehyde (DEAB) for 40 minutes at 37°C. Cells were centrifuged, washed and stained with 7-AAD (Biolegend) for dead cell exclusion. The fluorescence data were collected using either an LSR II flow cytometer (BD Bioscience) or a ZE5 cytometer (Biorad). To separate ALDH+ and ALDH− population, stained SUM-159 cells were sorted using a BD FACS Melody (BD Bioscience). The sorting gates were established using as negative controls the ALDEFLUOR-stained cells treated with DEAB. Data was processed and analyzed using FCS Express (De Novo Software).
8
Quantifying SARS-CoV-2 Spike Binding Affinity
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ExpiCHO cells were seeded into 50 ml Mini Bioreactor Centrifuge Tube (Corning) at 6 × 106 cells/ml in 5 ml ExpiCHO Expression medium (Life technology). Plasmids encoding SARS-CoV-2 Wuhan, Alpha (B.1.1.7), Delta (B.1.617.2) or Omicron (B.1.1.529) Spike protein (5 mg) were diluted in OptiPRO SFM (Life Technology) and mixed with Expifectamine CHO Reagent (Life Technology). After 1 min incubation at room temperature, transfection mixes were added to cell suspensions. Next, cells were incubated at 37°C 8% CO2 with an orbital shaking speed of 120 rpm for the following 48 hours.
Transiently transfected cells were harvested and washed with PBS, 1% BSA, 2 mM EDTA. Cells were counted, dispensed into round-bottom 96 well plates (Corning) and incubated with human IgG Fc-conjugated ACE2 serial dilutions (concentration range: 30’000 – 0.17 ng/ml) for 45 min at 4°C. After two washes, Alexa Fluor647 Goat Anti-Human IgG secondary Ab (1.5 mg/ml) (Jackson Immunoresearch) was added to the cells, which were then incubated for 30 min at 4°C in the dark. Cells were washed twice and resuspended in wash buffer for data acquisition at ZE5 cytometer (Biorad). To assess Spike protein expression level, an aliquot of each transfectant cell line was stained with 10 mg/ml of S2P6 antibody (Pinto et al., Science 2021) for 45 min at 4°C.
Transiently transfected cells were harvested and washed with PBS, 1% BSA, 2 mM EDTA. Cells were counted, dispensed into round-bottom 96 well plates (Corning) and incubated with human IgG Fc-conjugated ACE2 serial dilutions (concentration range: 30’000 – 0.17 ng/ml) for 45 min at 4°C. After two washes, Alexa Fluor647 Goat Anti-Human IgG secondary Ab (1.5 mg/ml) (Jackson Immunoresearch) was added to the cells, which were then incubated for 30 min at 4°C in the dark. Cells were washed twice and resuspended in wash buffer for data acquisition at ZE5 cytometer (Biorad). To assess Spike protein expression level, an aliquot of each transfectant cell line was stained with 10 mg/ml of S2P6 antibody (Pinto et al., Science 2021) for 45 min at 4°C.
9
Cell Cycle Analysis of A549 Cells
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A549 cells were seeded in six well plates. The day after, each drug was added in triplicate. Twenty-four hours later, adherent and floating cells were collected after trypsinization. The cells were then washed once with PBS followed by PBS containing 0.5% BSA, before being fixed in cold ethanol (70%). The cells were subsequently incubated in PBS containing 10 μg/ml propidium iodide (PI; Invitrogen Villebon-sur-Yvette, France) and 100 μg/ml RNase A (Invitrogen) overnight at 4°C. The data were acquired with a ZE5 cytometer (Biorad) (a minimum of 20 000 cells per sample were analyzed). The DNA content was quantified by using ModFit LT software (Verity Software House, Topsham, ME).
10
Quantifying SARS-CoV-2 Spike Protein Binding
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