For post infection flow cytometry, fixed iAT2s were either stained for cell surface expression of ACE2 (R&D, #AF933, 4-8 μg/2.5x106 cells) followed by donkey anti-goat IgG-AF647 (Invitrogen, #A21447) or were permeabilized with saponin buffer (Biolegend) then stained with SARS-CoV nucleoprotein (N) antibody (Rockland, #200-401-A50, 1:1000), followed by donkey anti-rabbit IgG-AF488 (Jackson ImmunoResearch, #711-545-152). Gating was based on either mock infected stained controls or infected, isotype-stained controls. Flow cytometry staining was quantified using a Stratedigm S1000EXI and analyzed with FlowJo v10.6.2 (FlowJo, Tree Star Inc). FACS plots shown represent single-cells based on forward-scatter/side-scatter gating.
Saponin buffer
Manufactured by BioLegend
Saponin buffer is a reagent used in various biological applications. It functions as a permeabilizing agent, allowing the passage of molecules across cell membranes. The buffer composition and concentration can be adjusted to suit specific experimental requirements.
Automatically generated - may contain errors
Lab products found in correlation
S1000exi, by Stratedigm (3 mentions)
Alexa fluor 488 anti rabbit igg, by Thermo Fisher Scientific (2 mentions)
Anti mouse igg1 alexafluor647, by Jackson ImmunoResearch (2 mentions)
Alexa fluor 488 anti mouse igg, by Thermo Fisher Scientific (2 mentions)
Anti human cd184 cxcr4 pe, by STEMCELL (2 mentions)
Anti human cd117 ckit apc, by Thermo Fisher Scientific (2 mentions)
Prism 8, by GraphPad (2 mentions)
Af933, by R&D Systems (1 mentions)
Donkey anti goat igg af647, by Thermo Fisher Scientific (1 mentions)
Goat anti mouse af647, by Jackson ImmunoResearch (1 mentions)
Click it plus edu alexa fluor 647 flow cytometry assay kit, by Thermo Fisher Scientific (1 mentions)
Donkey anti rabbit af488, by Jackson ImmunoResearch (1 mentions)
Permeabilization wash buffer, by BioLegend (1 mentions)
7 protocols using saponin buffer
1
Quantification of SARS-CoV-2 Infection in iAT2 Cells
2
Quantifying Endoderm Induction and Hepatocyte Markers
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Endoderm induction was quantified using anti-human CD184(CXCR4)-PE (StemCell Technologies) and anti-human CD117(CKIT)-APC (ThermoFisher Scientific) conjugated monoclonal antibodies.21 (link),25 (link) To quantify intracellular protein content, iHeps were fixed in 1.6% paraformaldehyde for 20 min at 37°C and then permeabilized in saponin buffer (BioLegend). Cells were first probed using AAT (Santa Cruz Biotechnologies), AFP (Abcam), ZAAT (a kind gift from Qiushi Tang and Chris Mueller), and then anti-mouse IgG1-AlexaFluor647 (Jackson ImmunoResearch), anti-rabbit IgG-AlexaFluor488 (ThermoFisher Scientific) and anti-mouse IgG-AlexaFluor488 (ThermoFisher Scientific) antibodies. Staining quantification was performed using BD FACSCalibur or Stratedigm S1000EXi and all gating was performed using isotype-stained controls. Data analysis was performed using FlowJo (Tree Star) and Prism8 (GraphPad) software.
3
Intracellular Flow Cytometry for β Cells
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For intracellular staining, the SC-β cell clusters were dissociated into single cells, fixed with 1.6% PFA at 37 °C for 20 min and permeabilized with 1:10 diluted saponin buffer (Biolegend, #421002) for 5 min. After washed with PBS, the cells were incubated with primary antibodies and fluorescence-conjugated secondary antibodies for 30 min at RT. The cells were then washed and resuspended in FACS buffer (PBS containing 0.5% BSA) for flow cytometry analysis by BD FACSVerse and Moflo Astrios 4 lasers. Data were analyzed by using FlowJo v10.
4
SARS-CoV-2 Nucleoprotein Antibody Staining
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After infection and fixation, HIOs were manually dissociated into single-cell suspension, permeabilized with saponin buffer (Biolegend), and stained with SARS-CoV-2 nucleoprotein (N) antibody (Rockland, #200-401-A50, 1:1000) for 30 min at RT, followed by an incubation with donkey anti-rabbit IgG-AF647 (Thermo Fisher A-31573). Gating was performed with mock-infected stained cells. Samples were run on a Stratedigm S1000EXI instrument and analyzed with the FlowJo v10.6.2 software (FlowJo, Tree Star Inc). Shown FACS plots represent single cells based on forward-scatter/side-scatter gating.
5
Multiparametric Flow Cytometry Analysis
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Cells were stained with cell surface antibodies against the following antigens: CXCR4 (PE-conjugated; Life Technologies, MHCXCR404), cKit (APC-conjugated; Life Technologies, CD11705). For intracellular staining, cells were fixed with 1.6% paraformaldehyde at 37°C for 20 min, then permeabilized in saponin buffer (BioLegend). Intracellular antigens were probed with the following antibodies: AAT (Santa Cruz, sc-59438), AFP (Abcam, ab169552), 2C1 (a kind gift from David Lomas and Elena Miranda), and/or BiP (Invitrogen, PA1-014A), then incubated with goat anti-mouse AF647 (Jackson ImmunoResearch, 115-605-003) and donkey anti-rabbit AF488 (Jackson ImmunoResearch, 711-545-152). To assess proliferation by EdU incorporation, cells were incubated for 24 h with 10 μM EdU, then stained using the Click-iT Plus EdU Alexa Fluor 647 Flow Cytometry Assay Kit (Life Technologies, C10635) as per manufacturer’s instructions. For all flow cytometry experiments, gating was based on isotype-stained controls. Stained cells were quantified using a Stratedigm S1000EON, and data were analyzed using FlowJo (Tree Star).
6
Quantifying Endoderm Induction and Hepatocyte Markers
7
Hepatocyte Specification Flow Cytometry
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Flow cytometry was performed at days 5, 14, and 25 of hepatocyte specification. For day 5, 3e5 cells were stained per condition, and the C-KIT antibody (Biolegend, #313206) and CXCR4 antibody (Invitrogen, #MHCXCR404) were used at a concentration of 5 μL per 1e6 cells. Staining was performed on ice for 30 minutes. For days 14 and 25, cells were fixed in 1.6% paraformaldehyde before staining. Primary antibodies for AAT (Santa Cruz, #sc-59438) and FOXA1 (Santa Cruz, #101058) were added at 1 : 100 ratio in Saponin Buffer (2% FBS, 1x Permeabilization Wash Buffer (Biolegend, #421002)) and incubated at room temperature for 30 minutes. Secondary antibodies for AAT (Jackson Immunoresearch, #115-605-205) and FOXA1 (Jackson Immunoresearch, #115-545-206) were added at a dilution of 1 : 500 and incubated at room temperature for 30 minutes. All samples were resuspended in PBS with 0.5% BSA for analysis.
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