anti sars nucleoprotein  (Rockland Immunochemicals)

 
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    Name:
    SARS CoV 2 Nucleocapsid N Protein Antibody
    Description:
    Anti SARS CoV 2 Nucleocapsid N Protein RABBIT Antibody 200 401 MS4 0100
    Catalog Number:
    200-401-MS4-0100
    Price:
    239.00
    Applications:
    ELISA , Western Blot
    Purity:
    This protein A purified antibody is directed against SARS Coronavirus 2 Nucleocapsid (N) protein. The product was purified from monospecific antiserum by protein A affinity purification. BLAST analysis was used to suggest reactivity with related Coronavirus proteins. Cross reactivity with homologues from other sources has not been determined.
    Immunogen:
    Anti-SARS-CoV-2 Nucleocapsid (N) Protein Antibody was produced by repeated immunizations with purified recombinant SARS-CoV-2 Nucleocapsid protein with C-terminal His-tag, derived from the transfected human HEK293 cells.
    Size:
    100 µg
    Product Aliases:
    rabbit anti-SARS CoV 2 Nucleocapsid Protein Antibody, N-protein antibody, SARS CoV2 antibody, 2019-nCoV, COVID-19, Severe acute respiratory syndrome antibody, Severe acute respiratory syndrome coronavirus 2
    Format:
    IgG
    Host:
    Rabbit
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    Structured Review

    Rockland Immunochemicals anti sars nucleoprotein
    SARS CoV 2 Nucleocapsid N Protein Antibody
    Anti SARS CoV 2 Nucleocapsid N Protein RABBIT Antibody 200 401 MS4 0100
    https://www.bioz.com/result/anti sars nucleoprotein/product/Rockland Immunochemicals
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti sars nucleoprotein - by Bioz Stars, 2021-09
    93/100 stars

    Images

    Related Articles

    Staining:

    Article Title: The in-vitro effect of famotidine on sars-cov-2 proteases and virus replication
    Article Snippet: .. At least 7–10 microscopic fields were imaged per well using a 10X objective lens, the number of cells positive for the SARS-CoV-2 N protein and the nuclear DAPI stain, were counted. ..

    Article Title: Mapping Systemic Inflammation and Antibody Responses in Multisystem Inflammatory Syndrome in Children (MIS-C)
    Article Snippet: .. Following fixation, the cells were washed, permeabilized with 0.1% Triton X-100, blocked in a 3% milk solution (American Bio) and stained with a monoclonal antibody to anti-SARS nucleoprotein ( ) and subsequently a goat anti-mouse IgG–HRP (Rockland Immunochemicals). ..

    Article Title: Comparative analysis reveals the species-specific genetic determinants of ACE2 required for SARS-CoV-2 entry
    Article Snippet: .. Analysis of SARS-CoV-2 infection by high-content imaging systemA549 cells were transduced with lentiviruses expressing the ACE2 of different species for 48 h. Cells were then infected with nCoV-SH01 (SARS-CoV-2) at an MOI of 1 for 1 h, washed three times with PBS, and incubated in 2% FBS culture medium for 48 h. Cell were then fixed for viral antigen staining with 4% paraformaldehyde in PBS, permeablized with 0.2% Triton X-100, and incubated with a rabbit polyclonal antibody against the SARS-CoV nucleocapsid protein (Rockland, 200-401-A50, 1μg/ml) and a mouse anti-FLAG M2 antibody (Sigma-Aldrich #1804, 1μg/ml) at 4°C overnight. ..

    Incubation:

    Article Title: A novel glucocorticoid and androgen receptor modulator reduces viral entry and innate immune inflammatory responses in the Syrian Hamster model of SARS-CoV-2
    Article Snippet: .. Primary antibodies were diluted to their optimized dilutions in tris-buffered saline and incubated on the tissue for 1 hour/antibody: Rabbit SARS nucleocapsid protein (SARS-CoV-2; Rockland; 1:500), goat ionized calcium binding adaptor molecule 1 (IBA1; Abcam; 1:50), goat angiotensin converting enzyme 2 (ACE2;R & D Systems; 1:500), rabbit transmembrane serine protease 2 (TMPRSS2; Abcam; 1:500), mouse interleukin 6 (IL-6; ThermoFisher; 1:500). ..

    Article Title: The DHODH Inhibitor PTC299 Arrests SARS-CoV-2 Replication and Suppresses Induction of Inflammatory Cytokines
    Article Snippet: .. After each step, the cells were washed three times in PBS, and then incubated for 1 hour at room temperature with a rabbit antibody directed against the SARS-CoV nucleoprotein (Rockland Immunochemicals, Gilbertsville, PA). ..

    Article Title: Comparative analysis reveals the species-specific genetic determinants of ACE2 required for SARS-CoV-2 entry
    Article Snippet: .. Analysis of SARS-CoV-2 infection by high-content imaging systemA549 cells were transduced with lentiviruses expressing the ACE2 of different species for 48 h. Cells were then infected with nCoV-SH01 (SARS-CoV-2) at an MOI of 1 for 1 h, washed three times with PBS, and incubated in 2% FBS culture medium for 48 h. Cell were then fixed for viral antigen staining with 4% paraformaldehyde in PBS, permeablized with 0.2% Triton X-100, and incubated with a rabbit polyclonal antibody against the SARS-CoV nucleocapsid protein (Rockland, 200-401-A50, 1μg/ml) and a mouse anti-FLAG M2 antibody (Sigma-Aldrich #1804, 1μg/ml) at 4°C overnight. ..

    Binding Assay:

    Article Title: A novel glucocorticoid and androgen receptor modulator reduces viral entry and innate immune inflammatory responses in the Syrian Hamster model of SARS-CoV-2
    Article Snippet: .. Primary antibodies were diluted to their optimized dilutions in tris-buffered saline and incubated on the tissue for 1 hour/antibody: Rabbit SARS nucleocapsid protein (SARS-CoV-2; Rockland; 1:500), goat ionized calcium binding adaptor molecule 1 (IBA1; Abcam; 1:50), goat angiotensin converting enzyme 2 (ACE2;R & D Systems; 1:500), rabbit transmembrane serine protease 2 (TMPRSS2; Abcam; 1:500), mouse interleukin 6 (IL-6; ThermoFisher; 1:500). ..

    Immunofluorescence:

    Article Title: Prolonged SARS-CoV-2 cell culture replication in respiratory samples from patients with severe COVID-19
    Article Snippet: .. SARS-CoV-2 CPE specificity was confirmed by immunofluorescence (shell-vial technique) by using a commercial anti-SARS-CoV-2 N protein (Rockland Immunochemicals, Inc., Limerick, PA, USA) as the primary antibody and a goat anti-rabbit IgG labelled with Alexafluor 488 (Abcam, Cambridge, UK) as the secondary antibody. ..

    Infection:

    Article Title: Comparative analysis reveals the species-specific genetic determinants of ACE2 required for SARS-CoV-2 entry
    Article Snippet: .. Analysis of SARS-CoV-2 infection by high-content imaging systemA549 cells were transduced with lentiviruses expressing the ACE2 of different species for 48 h. Cells were then infected with nCoV-SH01 (SARS-CoV-2) at an MOI of 1 for 1 h, washed three times with PBS, and incubated in 2% FBS culture medium for 48 h. Cell were then fixed for viral antigen staining with 4% paraformaldehyde in PBS, permeablized with 0.2% Triton X-100, and incubated with a rabbit polyclonal antibody against the SARS-CoV nucleocapsid protein (Rockland, 200-401-A50, 1μg/ml) and a mouse anti-FLAG M2 antibody (Sigma-Aldrich #1804, 1μg/ml) at 4°C overnight. ..

    Imaging:

    Article Title: Comparative analysis reveals the species-specific genetic determinants of ACE2 required for SARS-CoV-2 entry
    Article Snippet: .. Analysis of SARS-CoV-2 infection by high-content imaging systemA549 cells were transduced with lentiviruses expressing the ACE2 of different species for 48 h. Cells were then infected with nCoV-SH01 (SARS-CoV-2) at an MOI of 1 for 1 h, washed three times with PBS, and incubated in 2% FBS culture medium for 48 h. Cell were then fixed for viral antigen staining with 4% paraformaldehyde in PBS, permeablized with 0.2% Triton X-100, and incubated with a rabbit polyclonal antibody against the SARS-CoV nucleocapsid protein (Rockland, 200-401-A50, 1μg/ml) and a mouse anti-FLAG M2 antibody (Sigma-Aldrich #1804, 1μg/ml) at 4°C overnight. ..

    Transduction:

    Article Title: Comparative analysis reveals the species-specific genetic determinants of ACE2 required for SARS-CoV-2 entry
    Article Snippet: .. Analysis of SARS-CoV-2 infection by high-content imaging systemA549 cells were transduced with lentiviruses expressing the ACE2 of different species for 48 h. Cells were then infected with nCoV-SH01 (SARS-CoV-2) at an MOI of 1 for 1 h, washed three times with PBS, and incubated in 2% FBS culture medium for 48 h. Cell were then fixed for viral antigen staining with 4% paraformaldehyde in PBS, permeablized with 0.2% Triton X-100, and incubated with a rabbit polyclonal antibody against the SARS-CoV nucleocapsid protein (Rockland, 200-401-A50, 1μg/ml) and a mouse anti-FLAG M2 antibody (Sigma-Aldrich #1804, 1μg/ml) at 4°C overnight. ..

    Expressing:

    Article Title: Comparative analysis reveals the species-specific genetic determinants of ACE2 required for SARS-CoV-2 entry
    Article Snippet: .. Analysis of SARS-CoV-2 infection by high-content imaging systemA549 cells were transduced with lentiviruses expressing the ACE2 of different species for 48 h. Cells were then infected with nCoV-SH01 (SARS-CoV-2) at an MOI of 1 for 1 h, washed three times with PBS, and incubated in 2% FBS culture medium for 48 h. Cell were then fixed for viral antigen staining with 4% paraformaldehyde in PBS, permeablized with 0.2% Triton X-100, and incubated with a rabbit polyclonal antibody against the SARS-CoV nucleocapsid protein (Rockland, 200-401-A50, 1μg/ml) and a mouse anti-FLAG M2 antibody (Sigma-Aldrich #1804, 1μg/ml) at 4°C overnight. ..

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  • 95
    Rockland Immunochemicals sars nucleocapsid protein antibody
    <t>SARS-CoV-2</t> infects iAT2s in a dose- and time-dependent manner. (A) Schematic of the iAT2 directed differentiation protocol, in which robustly self-renewing iAT2s can be plated at air-liquid interface (ALI) for SARS-CoV-2 infections. “CK+DCI”=distal lung medium components detailed in the Materials and Methods. (B) Immunofluorescence images of viral nucleoprotein (N, green) of iAT2s infected with SARS-CoV-2 (MOI=5) at 1 and 4 days post infection (dpi), or (C) with increasing MOIs (0.5, 2.5, 5) shown at 1 dpi (20x, scale bar = 50μm). (D) Efficiency of iAT2 infections scored by representative FACS plots of SARS-CoV-2 N at 1 and 4 dpi (MOI 5) compared to mock; mean gated percentages +/- standard deviation for n=3 replicates are shown; results representative of 3 independent experiments. (E) RT-qPCR of viral N gene expression at 1 and 4 dpi using a range of low MOIs of an unpurified SARS-CoV-2 virus stock, n=3. (F, G) RT-qPCR of N gene expression at 1 and 4 dpi using a purified virus stock to infect with an MOI of 0.4 (F) or an MOI of 5 (G), n=3. Fold change expression compared to Mock [2 −ΔΔCt ] after 18S normalization is shown. (H) RT-qPCR of N gene expression of BU3 and SPC2 iAT2s at MOIs 0.04 and 0.4 at 1 dpi. (I) RT-qPCR of N gene expression at an MOI of 0.4 in iAT2s in alveolospheres and iAT2s at ALI at 2 dpi. (J) Viral titers were determined in apical washes and basolateral media at 1 and 4 dpi (n=5). (K) Mean percent fragmented nuclei in immunofluorescence images of infected iAT2s at 1 and 4 dpi (MOI 5; n=3). (L) Electron micrograph of infected iAT2s showing virions (arrow heads) intracellularly, including in a lamellar body and (M) extracellular virions around tubular myelin (N, arrow). Tubular myelin meshwork (inset from M) that forms upon secretion of pulmonary surfactant (size bar=200 nm). (O) Higher magnification of coronavirus virions at 4 dpi (MOI 5) (scale bar = 50 nm). All bars represent mean +/- standard deviation with biological replicates indicated for each panel. *p
    Sars Nucleocapsid Protein Antibody, supplied by Rockland Immunochemicals, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/sars nucleocapsid protein antibody/product/Rockland Immunochemicals
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    sars nucleocapsid protein antibody - by Bioz Stars, 2021-09
    95/100 stars
      Buy from Supplier

    Image Search Results


    SARS-CoV-2 infects iAT2s in a dose- and time-dependent manner. (A) Schematic of the iAT2 directed differentiation protocol, in which robustly self-renewing iAT2s can be plated at air-liquid interface (ALI) for SARS-CoV-2 infections. “CK+DCI”=distal lung medium components detailed in the Materials and Methods. (B) Immunofluorescence images of viral nucleoprotein (N, green) of iAT2s infected with SARS-CoV-2 (MOI=5) at 1 and 4 days post infection (dpi), or (C) with increasing MOIs (0.5, 2.5, 5) shown at 1 dpi (20x, scale bar = 50μm). (D) Efficiency of iAT2 infections scored by representative FACS plots of SARS-CoV-2 N at 1 and 4 dpi (MOI 5) compared to mock; mean gated percentages +/- standard deviation for n=3 replicates are shown; results representative of 3 independent experiments. (E) RT-qPCR of viral N gene expression at 1 and 4 dpi using a range of low MOIs of an unpurified SARS-CoV-2 virus stock, n=3. (F, G) RT-qPCR of N gene expression at 1 and 4 dpi using a purified virus stock to infect with an MOI of 0.4 (F) or an MOI of 5 (G), n=3. Fold change expression compared to Mock [2 −ΔΔCt ] after 18S normalization is shown. (H) RT-qPCR of N gene expression of BU3 and SPC2 iAT2s at MOIs 0.04 and 0.4 at 1 dpi. (I) RT-qPCR of N gene expression at an MOI of 0.4 in iAT2s in alveolospheres and iAT2s at ALI at 2 dpi. (J) Viral titers were determined in apical washes and basolateral media at 1 and 4 dpi (n=5). (K) Mean percent fragmented nuclei in immunofluorescence images of infected iAT2s at 1 and 4 dpi (MOI 5; n=3). (L) Electron micrograph of infected iAT2s showing virions (arrow heads) intracellularly, including in a lamellar body and (M) extracellular virions around tubular myelin (N, arrow). Tubular myelin meshwork (inset from M) that forms upon secretion of pulmonary surfactant (size bar=200 nm). (O) Higher magnification of coronavirus virions at 4 dpi (MOI 5) (scale bar = 50 nm). All bars represent mean +/- standard deviation with biological replicates indicated for each panel. *p

    Journal: bioRxiv

    Article Title: SARS-CoV-2 Infection of Pluripotent Stem Cell-derived Human Lung Alveolar Type 2 Cells Elicits a Rapid Epithelial-Intrinsic Inflammatory Response

    doi: 10.1101/2020.06.30.175695

    Figure Lengend Snippet: SARS-CoV-2 infects iAT2s in a dose- and time-dependent manner. (A) Schematic of the iAT2 directed differentiation protocol, in which robustly self-renewing iAT2s can be plated at air-liquid interface (ALI) for SARS-CoV-2 infections. “CK+DCI”=distal lung medium components detailed in the Materials and Methods. (B) Immunofluorescence images of viral nucleoprotein (N, green) of iAT2s infected with SARS-CoV-2 (MOI=5) at 1 and 4 days post infection (dpi), or (C) with increasing MOIs (0.5, 2.5, 5) shown at 1 dpi (20x, scale bar = 50μm). (D) Efficiency of iAT2 infections scored by representative FACS plots of SARS-CoV-2 N at 1 and 4 dpi (MOI 5) compared to mock; mean gated percentages +/- standard deviation for n=3 replicates are shown; results representative of 3 independent experiments. (E) RT-qPCR of viral N gene expression at 1 and 4 dpi using a range of low MOIs of an unpurified SARS-CoV-2 virus stock, n=3. (F, G) RT-qPCR of N gene expression at 1 and 4 dpi using a purified virus stock to infect with an MOI of 0.4 (F) or an MOI of 5 (G), n=3. Fold change expression compared to Mock [2 −ΔΔCt ] after 18S normalization is shown. (H) RT-qPCR of N gene expression of BU3 and SPC2 iAT2s at MOIs 0.04 and 0.4 at 1 dpi. (I) RT-qPCR of N gene expression at an MOI of 0.4 in iAT2s in alveolospheres and iAT2s at ALI at 2 dpi. (J) Viral titers were determined in apical washes and basolateral media at 1 and 4 dpi (n=5). (K) Mean percent fragmented nuclei in immunofluorescence images of infected iAT2s at 1 and 4 dpi (MOI 5; n=3). (L) Electron micrograph of infected iAT2s showing virions (arrow heads) intracellularly, including in a lamellar body and (M) extracellular virions around tubular myelin (N, arrow). Tubular myelin meshwork (inset from M) that forms upon secretion of pulmonary surfactant (size bar=200 nm). (O) Higher magnification of coronavirus virions at 4 dpi (MOI 5) (scale bar = 50 nm). All bars represent mean +/- standard deviation with biological replicates indicated for each panel. *p

    Article Snippet: Flow cytometry For post infection flow cytometry, fixed iAT2s were either stained for cell surface expression of ACE2 (R & D, #AF933, 4-8μg/2.5×106 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).

    Techniques: Immunofluorescence, Infection, FACS, Standard Deviation, Quantitative RT-PCR, Expressing, Purification

    Functional enrichment scores on lung-related Gene Ontology terms across SARS-CoV-2 infection models, Related to Figure 3 . Pre-ranked gene set enrichment analysis (FGSEA v.1.9.7) was performed on SARS-CoV-2 infected vs. mock-treated cells for 1 dpi iAT2s as well as other models systems like normal human bronchial epithelial cells (NHBE), A549-ACE2, Calu-3, and Vero E6 cells.

    Journal: bioRxiv

    Article Title: SARS-CoV-2 Infection of Pluripotent Stem Cell-derived Human Lung Alveolar Type 2 Cells Elicits a Rapid Epithelial-Intrinsic Inflammatory Response

    doi: 10.1101/2020.06.30.175695

    Figure Lengend Snippet: Functional enrichment scores on lung-related Gene Ontology terms across SARS-CoV-2 infection models, Related to Figure 3 . Pre-ranked gene set enrichment analysis (FGSEA v.1.9.7) was performed on SARS-CoV-2 infected vs. mock-treated cells for 1 dpi iAT2s as well as other models systems like normal human bronchial epithelial cells (NHBE), A549-ACE2, Calu-3, and Vero E6 cells.

    Article Snippet: Flow cytometry For post infection flow cytometry, fixed iAT2s were either stained for cell surface expression of ACE2 (R & D, #AF933, 4-8μg/2.5×106 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).

    Techniques: Functional Assay, Infection

    SARS-CoV-2 elicits transcriptomic changes in iAT2s that highlight epithelial-intrinsic inflammatory responses to infection. (A) Schematic of the iAT2 ALI samples (starting with Day 208 iAT2s) infected with SARS-CoV-2 (MOI 5) and collected at 1 and 4 dpi (mock collected 1 dpi) for bulk RNA sequencing (RNA-seq). (B) Principal component analysis (PCA) of iAT2 samples (n=3 biological replicates per condition) showing global transcriptomic variance (%) of PC1 and PC2 components. (C) Local regression (LOESS) plots of viral, AT2, NF-kB, and interferon (IFN) gene expression levels quantified by RNA-seq normalized expression (counts per million reads). (D) Gene set enrichment analysis (GSEA, Camera using Hallmark gene sets) of the top 10 upregulated gene sets in 1 dpi vs. mock or 4 dpi vs. 1 dpi conditions (black color indicates statistical significance; FDR

    Journal: bioRxiv

    Article Title: SARS-CoV-2 Infection of Pluripotent Stem Cell-derived Human Lung Alveolar Type 2 Cells Elicits a Rapid Epithelial-Intrinsic Inflammatory Response

    doi: 10.1101/2020.06.30.175695

    Figure Lengend Snippet: SARS-CoV-2 elicits transcriptomic changes in iAT2s that highlight epithelial-intrinsic inflammatory responses to infection. (A) Schematic of the iAT2 ALI samples (starting with Day 208 iAT2s) infected with SARS-CoV-2 (MOI 5) and collected at 1 and 4 dpi (mock collected 1 dpi) for bulk RNA sequencing (RNA-seq). (B) Principal component analysis (PCA) of iAT2 samples (n=3 biological replicates per condition) showing global transcriptomic variance (%) of PC1 and PC2 components. (C) Local regression (LOESS) plots of viral, AT2, NF-kB, and interferon (IFN) gene expression levels quantified by RNA-seq normalized expression (counts per million reads). (D) Gene set enrichment analysis (GSEA, Camera using Hallmark gene sets) of the top 10 upregulated gene sets in 1 dpi vs. mock or 4 dpi vs. 1 dpi conditions (black color indicates statistical significance; FDR

    Article Snippet: Flow cytometry For post infection flow cytometry, fixed iAT2s were either stained for cell surface expression of ACE2 (R & D, #AF933, 4-8μg/2.5×106 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).

    Techniques: Infection, RNA Sequencing Assay, Expressing

    Infection of iAT2s with SARS-CoV-2 prompts the loss of the lung AT2 program, activation of the NF-kB pathway, and delayed activation of IFN signaling. (A) Immunofluorescence staining of pro-surfactant protein C (pro-SFTPC) in tissue sections of non-COVID-19 and COVID-19 lungs with zoomed insets showing the typical cytoplasmic punctate appearance of lamellar body-localized pro-SFTPC. Arrow indicates AT2 hyperplasia (right panel), a typical and non-specific response to injury juxtaposed with regions that have a paucity of pro-SFTPC (middle panel). (B) COVID-19 decedent autopsied lung tissue sections, stained with H E. (C) Non-COVID-19 and COVID-19 decedent sections stained with cytokeratin AE1/AE3 (brown) showing early acute phase of diffuse alveolar damage with sloughed alveolar epithelium (200x magnification). (D) RT-qPCR of AT2 and (E) NFkB-related transcripts in iAT2s infected with SARS-CoV-2 (MOI 5; n=3; Fold change expression over “Mock”= 2 −ΔΔCt ) at 1 and 4 dpi. (F) Luminex analysis of apical washes and basolateral media collected from iAT2 ALI cultures (n=5). (G) RT-qPCR of interferon-stimulated genes (ISGs) in infected (MOI 5) iAT2s at 1 and 4 dpi (n=3). (H-I) RT-qPCR of N gene expression at 2 dpi (MOI 0.04) with (H) vehicle control, camostat (TMPRSS2 inhibitor), E-64d (cathepsin B/L inhibitor), or (I) remdesivir treatment, n=3. All bars represent mean +/- standard deviation.*p

    Journal: bioRxiv

    Article Title: SARS-CoV-2 Infection of Pluripotent Stem Cell-derived Human Lung Alveolar Type 2 Cells Elicits a Rapid Epithelial-Intrinsic Inflammatory Response

    doi: 10.1101/2020.06.30.175695

    Figure Lengend Snippet: Infection of iAT2s with SARS-CoV-2 prompts the loss of the lung AT2 program, activation of the NF-kB pathway, and delayed activation of IFN signaling. (A) Immunofluorescence staining of pro-surfactant protein C (pro-SFTPC) in tissue sections of non-COVID-19 and COVID-19 lungs with zoomed insets showing the typical cytoplasmic punctate appearance of lamellar body-localized pro-SFTPC. Arrow indicates AT2 hyperplasia (right panel), a typical and non-specific response to injury juxtaposed with regions that have a paucity of pro-SFTPC (middle panel). (B) COVID-19 decedent autopsied lung tissue sections, stained with H E. (C) Non-COVID-19 and COVID-19 decedent sections stained with cytokeratin AE1/AE3 (brown) showing early acute phase of diffuse alveolar damage with sloughed alveolar epithelium (200x magnification). (D) RT-qPCR of AT2 and (E) NFkB-related transcripts in iAT2s infected with SARS-CoV-2 (MOI 5; n=3; Fold change expression over “Mock”= 2 −ΔΔCt ) at 1 and 4 dpi. (F) Luminex analysis of apical washes and basolateral media collected from iAT2 ALI cultures (n=5). (G) RT-qPCR of interferon-stimulated genes (ISGs) in infected (MOI 5) iAT2s at 1 and 4 dpi (n=3). (H-I) RT-qPCR of N gene expression at 2 dpi (MOI 0.04) with (H) vehicle control, camostat (TMPRSS2 inhibitor), E-64d (cathepsin B/L inhibitor), or (I) remdesivir treatment, n=3. All bars represent mean +/- standard deviation.*p

    Article Snippet: Flow cytometry For post infection flow cytometry, fixed iAT2s were either stained for cell surface expression of ACE2 (R & D, #AF933, 4-8μg/2.5×106 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).

    Techniques: Infection, Activation Assay, Immunofluorescence, Staining, Quantitative RT-PCR, Expressing, Luminex, Standard Deviation

    iPSC-derived alveolar epithelial type 2 cells (iAT2s) express functional SARS-CoV-2 entry factors ACE2 and TMPRSS2. (A-B) iAT2s, carrying a tdTomato reporter targeted to the endogenous SFTPC locus by gene editing (SPC2 line), can be serially passaged while maintaining > 90% SFTPC tdTomato+ expression in 3D sphere cultures (Day 160 of differentiation, passage 8 shown; single cell RNA sequencing profile provided in Figure S1 ). (C, D) Single cell RNA sequencing data of iAT2s (SPC2 line at Day 114 of differentiation) visualized in SPRING plots (Weinreb et al., 2018) based on reanalysis of a dataset previously published in ( Hurley et al., 2020 ) showing expression of (C) SFTPC as well as (D) an 8-gene benchmark of AT2 cell differentiation ( SFTPC, CLDN18, LAMP3, SFTPB, SFTPD, NAPSA, SLC34A2, CXCL8 as characterized in ( Hurley et al., 2020 )). (E) iAT2s (RUES2 line) express ACE2 and TMPRSS2 transcripts at comparable levels to purified primary adult human lung AT2s (day 15 PLP=primordial lung progenitors derived from pluripotent stem cells at day 15 of differentiation, Early HFL=primary early human fetal lung alveolar epithelium at 16-17.5 weeks gestation; late HFL=alveolar epithelium at 20-21 weeks gestation, and adult AT2s=adult alveolar epithelial type 2 cells from 3 different individuals freshly sorted using the antibody HTII-280.adult AT2s; primary sample adult and fetal procurement described in detail in ( 9 )). (F-H) iAT2s (SPC2 line) cultured at air-liquid interface (ALI) (F) express ACE2 protein, as observed by flow cytometry, n=9 (G; additional scRNA-seq profiling in Figure S1 ), which is apically localized, as observed by immunofluorescence staining (scale bar = 10 μm) (H). (I-J) iAT2s infected with a GFP-expressing lentivirus pseudotyped with either VSVG or SARS-CoV-2 Spike envelopes, n=3. *p

    Journal: bioRxiv

    Article Title: SARS-CoV-2 Infection of Pluripotent Stem Cell-derived Human Lung Alveolar Type 2 Cells Elicits a Rapid Epithelial-Intrinsic Inflammatory Response

    doi: 10.1101/2020.06.30.175695

    Figure Lengend Snippet: iPSC-derived alveolar epithelial type 2 cells (iAT2s) express functional SARS-CoV-2 entry factors ACE2 and TMPRSS2. (A-B) iAT2s, carrying a tdTomato reporter targeted to the endogenous SFTPC locus by gene editing (SPC2 line), can be serially passaged while maintaining > 90% SFTPC tdTomato+ expression in 3D sphere cultures (Day 160 of differentiation, passage 8 shown; single cell RNA sequencing profile provided in Figure S1 ). (C, D) Single cell RNA sequencing data of iAT2s (SPC2 line at Day 114 of differentiation) visualized in SPRING plots (Weinreb et al., 2018) based on reanalysis of a dataset previously published in ( Hurley et al., 2020 ) showing expression of (C) SFTPC as well as (D) an 8-gene benchmark of AT2 cell differentiation ( SFTPC, CLDN18, LAMP3, SFTPB, SFTPD, NAPSA, SLC34A2, CXCL8 as characterized in ( Hurley et al., 2020 )). (E) iAT2s (RUES2 line) express ACE2 and TMPRSS2 transcripts at comparable levels to purified primary adult human lung AT2s (day 15 PLP=primordial lung progenitors derived from pluripotent stem cells at day 15 of differentiation, Early HFL=primary early human fetal lung alveolar epithelium at 16-17.5 weeks gestation; late HFL=alveolar epithelium at 20-21 weeks gestation, and adult AT2s=adult alveolar epithelial type 2 cells from 3 different individuals freshly sorted using the antibody HTII-280.adult AT2s; primary sample adult and fetal procurement described in detail in ( 9 )). (F-H) iAT2s (SPC2 line) cultured at air-liquid interface (ALI) (F) express ACE2 protein, as observed by flow cytometry, n=9 (G; additional scRNA-seq profiling in Figure S1 ), which is apically localized, as observed by immunofluorescence staining (scale bar = 10 μm) (H). (I-J) iAT2s infected with a GFP-expressing lentivirus pseudotyped with either VSVG or SARS-CoV-2 Spike envelopes, n=3. *p

    Article Snippet: Flow cytometry For post infection flow cytometry, fixed iAT2s were either stained for cell surface expression of ACE2 (R & D, #AF933, 4-8μg/2.5×106 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).

    Techniques: Derivative Assay, Functional Assay, Expressing, RNA Sequencing Assay, Cell Differentiation, Purification, Plasmid Purification, Cell Culture, Flow Cytometry, Immunofluorescence, Staining, Infection

    RT-qPCR of interferon stimulated genes (ISGs) in iAT2s infected with SARS-CoV-2, Related to Figure 4 . (A) Expression of ISGs in SARS-CoV-2-infected iAT2s (MOI 0.4) at 1 and 4 dpi. (B) Expression of ISGs and IFNs in SARS-CoV-2-infected iAT2s (MOI 0.4) at 4 dpi compared to 24h poly(I:C) (10 μg/mL) stimulation transfected by Oligofectamine vs. control and 24h recombinant human IFNb (rhIFNb, 10 ng/mL) vs. control. All bars represent mean +/- standard deviation, n=3. *p

    Journal: bioRxiv

    Article Title: SARS-CoV-2 Infection of Pluripotent Stem Cell-derived Human Lung Alveolar Type 2 Cells Elicits a Rapid Epithelial-Intrinsic Inflammatory Response

    doi: 10.1101/2020.06.30.175695

    Figure Lengend Snippet: RT-qPCR of interferon stimulated genes (ISGs) in iAT2s infected with SARS-CoV-2, Related to Figure 4 . (A) Expression of ISGs in SARS-CoV-2-infected iAT2s (MOI 0.4) at 1 and 4 dpi. (B) Expression of ISGs and IFNs in SARS-CoV-2-infected iAT2s (MOI 0.4) at 4 dpi compared to 24h poly(I:C) (10 μg/mL) stimulation transfected by Oligofectamine vs. control and 24h recombinant human IFNb (rhIFNb, 10 ng/mL) vs. control. All bars represent mean +/- standard deviation, n=3. *p

    Article Snippet: Flow cytometry For post infection flow cytometry, fixed iAT2s were either stained for cell surface expression of ACE2 (R & D, #AF933, 4-8μg/2.5×106 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).

    Techniques: Quantitative RT-PCR, Infection, Expressing, Transfection, Recombinant, Standard Deviation

    iPSC-derived airway is permissive to SARS-CoV-2 infection with time-dependent restriction in viral growth A) Schematic of the protocol to iPSC-airway with SARS-CoV-2. B-E) Immunofluorescence and quantification of viral nucleoprotein+ (SARS-CoV-2 N, green) cells in BU3 NGPT (B-C) and 1566 (D-E) cells at 1 dpi (40x, scale bar=100μm). BU3 NGPT mean nucleoprotein+ cells =6.87%±0.548 (SEM). 1566 mean nucleoprotein+ cells = 11.21% ± 1.075 (SEM). F) Confocal immunofluorescence microscopy of BU3 NGPT with antibodies against SARS-CoV-2 N positive (green) cells and α-TUBULIN (red). Nuclei are stained with DAPI (blue). G) Immunofluorescence of infected iPSC airway (BU3 NGPT) at 1 and 3 dpi, labeled with anti-SARS-CoV-2 N antibody and nuclei labeled with DAPI (20x, scale bar =100um). H) RT-qPCR of viral N gene expression of iPSC-airway (BU3 NGPT) at 1 and 3 dpi (n = 3 Transwells per sample). Fold change expression compared to mock [2 −ddCt ] after 18S normalization is shown. I) Percent of fragmented nuclei detected at 1 and 3 dpi in iPSC-airway (BU3 NGPT)(1 dpi=mean 0.69±0.12% (SEM) and 3 dpi (mean 2.14±0.32 (SEM)). J) Viral titers from apical washes and basolateral media at 1 and 3 dpi from iPSC-airway (BU3 NGPT) compared to mock. K) Transmission electron micrograph of SARS-CoV-2 infected iPSC-airway (1566) at 1 dpi. Blue arrows indicate viral particles (Scale bar= 200nm, 50nm in enclosed box)

    Journal: bioRxiv

    Article Title: Human airway lineages derived from pluripotent stem cells reveal the epithelial responses to SARS-CoV-2 infection

    doi: 10.1101/2021.07.06.451340

    Figure Lengend Snippet: iPSC-derived airway is permissive to SARS-CoV-2 infection with time-dependent restriction in viral growth A) Schematic of the protocol to iPSC-airway with SARS-CoV-2. B-E) Immunofluorescence and quantification of viral nucleoprotein+ (SARS-CoV-2 N, green) cells in BU3 NGPT (B-C) and 1566 (D-E) cells at 1 dpi (40x, scale bar=100μm). BU3 NGPT mean nucleoprotein+ cells =6.87%±0.548 (SEM). 1566 mean nucleoprotein+ cells = 11.21% ± 1.075 (SEM). F) Confocal immunofluorescence microscopy of BU3 NGPT with antibodies against SARS-CoV-2 N positive (green) cells and α-TUBULIN (red). Nuclei are stained with DAPI (blue). G) Immunofluorescence of infected iPSC airway (BU3 NGPT) at 1 and 3 dpi, labeled with anti-SARS-CoV-2 N antibody and nuclei labeled with DAPI (20x, scale bar =100um). H) RT-qPCR of viral N gene expression of iPSC-airway (BU3 NGPT) at 1 and 3 dpi (n = 3 Transwells per sample). Fold change expression compared to mock [2 −ddCt ] after 18S normalization is shown. I) Percent of fragmented nuclei detected at 1 and 3 dpi in iPSC-airway (BU3 NGPT)(1 dpi=mean 0.69±0.12% (SEM) and 3 dpi (mean 2.14±0.32 (SEM)). J) Viral titers from apical washes and basolateral media at 1 and 3 dpi from iPSC-airway (BU3 NGPT) compared to mock. K) Transmission electron micrograph of SARS-CoV-2 infected iPSC-airway (1566) at 1 dpi. Blue arrows indicate viral particles (Scale bar= 200nm, 50nm in enclosed box)

    Article Snippet: The antibodies used were; anti-SARS-CoV nucleoprotein (N) antibody (rabbit polyclonal, 1:2500, Rockland Immunochemicals, Cat #200-401-A50), anti-α-TUBULIN antibody (mouse monoclonal, 1:500 sigma cat# T6199), and anti-MUC5B antibody (mouse monoclonal, Santa Cruz Biotechnology, 1:500 cat#SC-39395-2).

    Techniques: Derivative Assay, Infection, Immunofluorescence, Microscopy, Staining, Labeling, Quantitative RT-PCR, Expressing, Transmission Assay

    Transcriptomic analysis of SARS-CoV-2 infected iPSC-airway shows robust interferon response A) Schematic of the experimental design to compare SARS-CoV-2 infected iPSC-airway samples (BU3 NGPT) at 1 and 3 dpi to mock controls by RNA-seq (n=3 Transwells). Figures 4B-I are analyses of this experiment. B) PCA comparing PC1 vs PC2 of the samples described in A. C) The top 50 DEGs ranked by fold change (FDR

    Journal: bioRxiv

    Article Title: Human airway lineages derived from pluripotent stem cells reveal the epithelial responses to SARS-CoV-2 infection

    doi: 10.1101/2021.07.06.451340

    Figure Lengend Snippet: Transcriptomic analysis of SARS-CoV-2 infected iPSC-airway shows robust interferon response A) Schematic of the experimental design to compare SARS-CoV-2 infected iPSC-airway samples (BU3 NGPT) at 1 and 3 dpi to mock controls by RNA-seq (n=3 Transwells). Figures 4B-I are analyses of this experiment. B) PCA comparing PC1 vs PC2 of the samples described in A. C) The top 50 DEGs ranked by fold change (FDR

    Article Snippet: The antibodies used were; anti-SARS-CoV nucleoprotein (N) antibody (rabbit polyclonal, 1:2500, Rockland Immunochemicals, Cat #200-401-A50), anti-α-TUBULIN antibody (mouse monoclonal, 1:500 sigma cat# T6199), and anti-MUC5B antibody (mouse monoclonal, Santa Cruz Biotechnology, 1:500 cat#SC-39395-2).

    Techniques: Infection, RNA Sequencing Assay

    iPSC-airway infected with SARS-CoV-2 secrete inflammatory cytokines and chemokines and detects antiviral drug effects. A) Luminex analysis of apical washes and basolateral media collected from iPSC-airway (BU3 NGPT, cultures (n=3 Transwells). B) RT-qPCR of select genes iPSC-airway (BU3 NGPT) infected with SARS-CoV-2 (MOI 4) and harvested at 1 and 3 dpi with their respective mock-infected samples (n=3 Transwells). Fold change expression compared to mock [2 −ddCt ] after 18S normalization is shown. RT-qPCR of N gene expression of mock-infected and SARS-CoV-2 infected (MOI 0.04) iPSC-airway (BU3 NGPT) at 2 dpi pretreated with C) vehicle control (DMSO) or remdesivir (10 μM) or D) vehicle control (DMSO) or camostat (TMPRSS2 inhibitor, 1, 10, 100 μM, as indicated (n= 3 Transwells for both C and D).

    Journal: bioRxiv

    Article Title: Human airway lineages derived from pluripotent stem cells reveal the epithelial responses to SARS-CoV-2 infection

    doi: 10.1101/2021.07.06.451340

    Figure Lengend Snippet: iPSC-airway infected with SARS-CoV-2 secrete inflammatory cytokines and chemokines and detects antiviral drug effects. A) Luminex analysis of apical washes and basolateral media collected from iPSC-airway (BU3 NGPT, cultures (n=3 Transwells). B) RT-qPCR of select genes iPSC-airway (BU3 NGPT) infected with SARS-CoV-2 (MOI 4) and harvested at 1 and 3 dpi with their respective mock-infected samples (n=3 Transwells). Fold change expression compared to mock [2 −ddCt ] after 18S normalization is shown. RT-qPCR of N gene expression of mock-infected and SARS-CoV-2 infected (MOI 0.04) iPSC-airway (BU3 NGPT) at 2 dpi pretreated with C) vehicle control (DMSO) or remdesivir (10 μM) or D) vehicle control (DMSO) or camostat (TMPRSS2 inhibitor, 1, 10, 100 μM, as indicated (n= 3 Transwells for both C and D).

    Article Snippet: The antibodies used were; anti-SARS-CoV nucleoprotein (N) antibody (rabbit polyclonal, 1:2500, Rockland Immunochemicals, Cat #200-401-A50), anti-α-TUBULIN antibody (mouse monoclonal, 1:500 sigma cat# T6199), and anti-MUC5B antibody (mouse monoclonal, Santa Cruz Biotechnology, 1:500 cat#SC-39395-2).

    Techniques: Infection, Luminex, Quantitative RT-PCR, Expressing

    iPSC-derived airway cells express SARS-CoV-2 entry factors ACE2 and TMPRSS2 A) Schematic of the 6-stage differentiation protocol of generating iPSC-airway. B) Immunofluorescence analysis of BU3 NGPT iPSC-airway stained with anti-α-TUBULIN and MUC5AC (scale bar = 100 μm). Nuclei are stained with HOESCHT (blue). C) Immunofluorescence analysis of 1566 iPSC airway, stained with anti-α-TUBULIN and MUC5B (scale bar = 100μm). Nuclei are stained with DAPI (blue). D-G) scRNA-seq analysis of HBEC 40 , iPSC-airway (BU3 NGPT) 40 , and freshly isolated uncultured lung epithelia 42 . D) Violin plots of ACE2 and TMPRSS2 expression. E) The percentage of ACE2 and TMPRSS2 positive cells in each dataset 40 . F) Violin plots of ACE2 and TMPRSS2 expression by cellular type in each dataset. G) Comparison of the percentage of ACE2 and TMPRSS2 positive secretory, multiciliated, and basal cells in each dataset. H-I) Immunohistochemistry staining showing the localization of ACE2 (DAB), α-TUBULIN (purple, left panels) MUCIN (yellow, right panels) in iPSC-derived airway (BU3 NGPT) counterstained with hematoxylin (20x, scale bar =50μm). Lower panels are zoomed-in images of the black box in the upper panels. The red arrows indicated cells co-expressing ACE2/ α-TUBULIN (left) and ACE2/MUCIN (right) (scale bar =25μm).

    Journal: bioRxiv

    Article Title: Human airway lineages derived from pluripotent stem cells reveal the epithelial responses to SARS-CoV-2 infection

    doi: 10.1101/2021.07.06.451340

    Figure Lengend Snippet: iPSC-derived airway cells express SARS-CoV-2 entry factors ACE2 and TMPRSS2 A) Schematic of the 6-stage differentiation protocol of generating iPSC-airway. B) Immunofluorescence analysis of BU3 NGPT iPSC-airway stained with anti-α-TUBULIN and MUC5AC (scale bar = 100 μm). Nuclei are stained with HOESCHT (blue). C) Immunofluorescence analysis of 1566 iPSC airway, stained with anti-α-TUBULIN and MUC5B (scale bar = 100μm). Nuclei are stained with DAPI (blue). D-G) scRNA-seq analysis of HBEC 40 , iPSC-airway (BU3 NGPT) 40 , and freshly isolated uncultured lung epithelia 42 . D) Violin plots of ACE2 and TMPRSS2 expression. E) The percentage of ACE2 and TMPRSS2 positive cells in each dataset 40 . F) Violin plots of ACE2 and TMPRSS2 expression by cellular type in each dataset. G) Comparison of the percentage of ACE2 and TMPRSS2 positive secretory, multiciliated, and basal cells in each dataset. H-I) Immunohistochemistry staining showing the localization of ACE2 (DAB), α-TUBULIN (purple, left panels) MUCIN (yellow, right panels) in iPSC-derived airway (BU3 NGPT) counterstained with hematoxylin (20x, scale bar =50μm). Lower panels are zoomed-in images of the black box in the upper panels. The red arrows indicated cells co-expressing ACE2/ α-TUBULIN (left) and ACE2/MUCIN (right) (scale bar =25μm).

    Article Snippet: The antibodies used were; anti-SARS-CoV nucleoprotein (N) antibody (rabbit polyclonal, 1:2500, Rockland Immunochemicals, Cat #200-401-A50), anti-α-TUBULIN antibody (mouse monoclonal, 1:500 sigma cat# T6199), and anti-MUC5B antibody (mouse monoclonal, Santa Cruz Biotechnology, 1:500 cat#SC-39395-2).

    Techniques: Derivative Assay, Immunofluorescence, Staining, Isolation, Expressing, Immunohistochemistry

    (related to Figure 3 ). Transcriptomic analysis of SARS-CoV-2 infected iPSC-airway shows a rapid and robust interferon response A) Unsupervised hierarchical clustered heatmaps of differentially expressed genes (DEGs; −log2FC) between mock-infected and SARS-CoV-2 infected iPSC-airway (BU3 NGPT) samples and samples at 3 dpi. B) Gene set enrichment analysis (GSEA) of the top ten upregulated gene sets in mock versus SARS-CoV-2 (infected iPSC-airway (1566) at 1 dpi. C) Unsupervised hierarchical clustered heatmaps of differentially expressed genes (DEGs; −log2FC) between mock versus SARS-CoV-2 infected iPSC-airway (1566) samples at 1 dpi. D) Volcano plots of differentially expressed genes in mock versus SARS-CoV-2 infected iPSC-airway (1566) at 1 dpi. E) Local regression (LOESS) plots of viral, interferon and ISG, and inflammatory gene expression levels quantified by RNA-seq normalized expression (counts per million reads) for iPSC-airway (BU3 NGPT). F) RT-qPCR of IFNL1, IFIT1, and IL6 in iPSC-airway (BU3 NGPT) that have been mock infected or infected with SARS-CoV-2 (MOI 4) at 1 dpi compared to poly(I:C) transfection (10 μg/mL), or treatment with recombinant human IFNβ(10 ug/mL) for 24 hours. Fold change expression compared to mock [2 −ddCt ] after 18S normalization is shown.

    Journal: bioRxiv

    Article Title: Human airway lineages derived from pluripotent stem cells reveal the epithelial responses to SARS-CoV-2 infection

    doi: 10.1101/2021.07.06.451340

    Figure Lengend Snippet: (related to Figure 3 ). Transcriptomic analysis of SARS-CoV-2 infected iPSC-airway shows a rapid and robust interferon response A) Unsupervised hierarchical clustered heatmaps of differentially expressed genes (DEGs; −log2FC) between mock-infected and SARS-CoV-2 infected iPSC-airway (BU3 NGPT) samples and samples at 3 dpi. B) Gene set enrichment analysis (GSEA) of the top ten upregulated gene sets in mock versus SARS-CoV-2 (infected iPSC-airway (1566) at 1 dpi. C) Unsupervised hierarchical clustered heatmaps of differentially expressed genes (DEGs; −log2FC) between mock versus SARS-CoV-2 infected iPSC-airway (1566) samples at 1 dpi. D) Volcano plots of differentially expressed genes in mock versus SARS-CoV-2 infected iPSC-airway (1566) at 1 dpi. E) Local regression (LOESS) plots of viral, interferon and ISG, and inflammatory gene expression levels quantified by RNA-seq normalized expression (counts per million reads) for iPSC-airway (BU3 NGPT). F) RT-qPCR of IFNL1, IFIT1, and IL6 in iPSC-airway (BU3 NGPT) that have been mock infected or infected with SARS-CoV-2 (MOI 4) at 1 dpi compared to poly(I:C) transfection (10 μg/mL), or treatment with recombinant human IFNβ(10 ug/mL) for 24 hours. Fold change expression compared to mock [2 −ddCt ] after 18S normalization is shown.

    Article Snippet: The antibodies used were; anti-SARS-CoV nucleoprotein (N) antibody (rabbit polyclonal, 1:2500, Rockland Immunochemicals, Cat #200-401-A50), anti-α-TUBULIN antibody (mouse monoclonal, 1:500 sigma cat# T6199), and anti-MUC5B antibody (mouse monoclonal, Santa Cruz Biotechnology, 1:500 cat#SC-39395-2).

    Techniques: Infection, Expressing, RNA Sequencing Assay, Quantitative RT-PCR, Transfection, Recombinant

    Transmission Electron Microscopy of SARS-CoV-2-Infected Colonic and Proximal HIOS Representative electron micrographs of (A) proximal HIO enterocytes infected with SARS-CoV-2 showing large numbers of coronavirus particles adjacent to the cell surface (inset, white arrowheads), (B) proximal HIO cytoplasm with budding virions (inset, white arrowheads), (C) infected colonic HIOs with intracellular replication complexes (white arrowheads) and viral particles within a single membrane vesicle (inset), and (D) cytoplasmic regions of viral replication with characteristic horseshoe structures and budding virions (inset). Scale bars, 500 nm (A, C, D large images), 200 nm (B, large image), or 125 nm (all insets); images representative of n = 3 replicate directed differentiation experiments. See also Figure S2 .

    Journal: Stem Cell Reports

    Article Title: Human Pluripotent Stem Cell-Derived Intestinal Organoids Model SARS-CoV-2 Infection Revealing a Common Epithelial Inflammatory Response

    doi: 10.1016/j.stemcr.2021.02.019

    Figure Lengend Snippet: Transmission Electron Microscopy of SARS-CoV-2-Infected Colonic and Proximal HIOS Representative electron micrographs of (A) proximal HIO enterocytes infected with SARS-CoV-2 showing large numbers of coronavirus particles adjacent to the cell surface (inset, white arrowheads), (B) proximal HIO cytoplasm with budding virions (inset, white arrowheads), (C) infected colonic HIOs with intracellular replication complexes (white arrowheads) and viral particles within a single membrane vesicle (inset), and (D) cytoplasmic regions of viral replication with characteristic horseshoe structures and budding virions (inset). Scale bars, 500 nm (A, C, D large images), 200 nm (B, large image), or 125 nm (all insets); images representative of n = 3 replicate directed differentiation experiments. See also Figure S2 .

    Article Snippet: Flow CytometryAfter 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).

    Techniques: Transmission Assay, Electron Microscopy, Infection

    Transcriptomic Response to SARS-CoV-2 Infection in Colonic and Proximal HIOs (A) Experimental schematic of RNA-seq experiment, with n = 3 replicate directed differentiations per condition tested (colonic/proximal HIOs, 1 and 4 dpi with corresponding mock controls). (B) PCA plot of mock and infected proximal and distal samples colored by tissue type, showing global transcriptomic variance between proximal and colonic HIOs. (C) LOESS plots of key viral genes in mock (left) and infected (right) HIOs at 1 and 4 dpi. (D) Unsupervised hierarchical clustered heatmap of differential gene expression of a subset of interferon-related genes as plotted by log fold change in HIOs at 1 and 4 dpi versus mock. (E) GSEA (using hallmark gene sets) of top differentially expressed gene sets in colonic and proximal HIOs 1 and 4 dpi versus mock, compared with previously published SARS-CoV-2-related datasets, including ASC-derived intestinal organoids, Calu-3 cells, and iPSC-derived alveolar type 2 cells cultured at air-liquid interface. Statistical significance (p

    Journal: Stem Cell Reports

    Article Title: Human Pluripotent Stem Cell-Derived Intestinal Organoids Model SARS-CoV-2 Infection Revealing a Common Epithelial Inflammatory Response

    doi: 10.1016/j.stemcr.2021.02.019

    Figure Lengend Snippet: Transcriptomic Response to SARS-CoV-2 Infection in Colonic and Proximal HIOs (A) Experimental schematic of RNA-seq experiment, with n = 3 replicate directed differentiations per condition tested (colonic/proximal HIOs, 1 and 4 dpi with corresponding mock controls). (B) PCA plot of mock and infected proximal and distal samples colored by tissue type, showing global transcriptomic variance between proximal and colonic HIOs. (C) LOESS plots of key viral genes in mock (left) and infected (right) HIOs at 1 and 4 dpi. (D) Unsupervised hierarchical clustered heatmap of differential gene expression of a subset of interferon-related genes as plotted by log fold change in HIOs at 1 and 4 dpi versus mock. (E) GSEA (using hallmark gene sets) of top differentially expressed gene sets in colonic and proximal HIOs 1 and 4 dpi versus mock, compared with previously published SARS-CoV-2-related datasets, including ASC-derived intestinal organoids, Calu-3 cells, and iPSC-derived alveolar type 2 cells cultured at air-liquid interface. Statistical significance (p

    Article Snippet: Flow CytometryAfter 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).

    Techniques: Infection, RNA Sequencing Assay, Expressing, Derivative Assay, Cell Culture

    SARS-CoV-2 Infects iPSC-Derived Human Intestinal Organoids (A) Experimental schematic for directed differentiation of BU1CG iPSC into HIOs and SARS-CoV-2 infection. (B) Representative confocal images of colonic BU1CG HIOs. Whole-mounted mock- and SARS-CoV-2-infected colonic HIOs at 1 and 4 dpi stained for SARS-CoV-2 N and GFP (CDX2) (scale bar,50 μm). (C) Representative immunofluorescent confocal micrographs of proximally patterned BU1CG. Whole-mounted mock- and SARS-CoV2-infected proximal (small intestinal) HIOs at 1 and 4 dpi stained for SARS-CoV-2 N and GFP (CDX2) (scale bar,50 μm, images representative of n = 3 replicate directed differentiations). (D) qRT-PCR for two sequences of the SARS-CoV-2 N gene in both proximal and colonic HIOs 1 and 3 dpi (2 −ΔΔCT , technical duplicates normalized to mock, GAPDH , n = 3 independent directed differentiations, error bars represent the SD, statistical significance where indicated determined by unpaired Student's t test, ∗ p

    Journal: Stem Cell Reports

    Article Title: Human Pluripotent Stem Cell-Derived Intestinal Organoids Model SARS-CoV-2 Infection Revealing a Common Epithelial Inflammatory Response

    doi: 10.1016/j.stemcr.2021.02.019

    Figure Lengend Snippet: SARS-CoV-2 Infects iPSC-Derived Human Intestinal Organoids (A) Experimental schematic for directed differentiation of BU1CG iPSC into HIOs and SARS-CoV-2 infection. (B) Representative confocal images of colonic BU1CG HIOs. Whole-mounted mock- and SARS-CoV-2-infected colonic HIOs at 1 and 4 dpi stained for SARS-CoV-2 N and GFP (CDX2) (scale bar,50 μm). (C) Representative immunofluorescent confocal micrographs of proximally patterned BU1CG. Whole-mounted mock- and SARS-CoV2-infected proximal (small intestinal) HIOs at 1 and 4 dpi stained for SARS-CoV-2 N and GFP (CDX2) (scale bar,50 μm, images representative of n = 3 replicate directed differentiations). (D) qRT-PCR for two sequences of the SARS-CoV-2 N gene in both proximal and colonic HIOs 1 and 3 dpi (2 −ΔΔCT , technical duplicates normalized to mock, GAPDH , n = 3 independent directed differentiations, error bars represent the SD, statistical significance where indicated determined by unpaired Student's t test, ∗ p

    Article Snippet: Flow CytometryAfter 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).

    Techniques: Derivative Assay, Infection, Staining, Quantitative RT-PCR

    HIOs Maintain Their Regional Identity after SARS-CoV-2 Infection (A) qRT-PCR for phenotypic intestinal markers at 1 and 3 dpi colonic HIOs (2 −ΔΔCT , technical duplicates normalized to GAPDH , n = 3 independent directed differentiation experiments, error bars represent the SD, statistical significance where indicated determined by unpaired Student's t test, ∗∗ = p

    Journal: Stem Cell Reports

    Article Title: Human Pluripotent Stem Cell-Derived Intestinal Organoids Model SARS-CoV-2 Infection Revealing a Common Epithelial Inflammatory Response

    doi: 10.1016/j.stemcr.2021.02.019

    Figure Lengend Snippet: HIOs Maintain Their Regional Identity after SARS-CoV-2 Infection (A) qRT-PCR for phenotypic intestinal markers at 1 and 3 dpi colonic HIOs (2 −ΔΔCT , technical duplicates normalized to GAPDH , n = 3 independent directed differentiation experiments, error bars represent the SD, statistical significance where indicated determined by unpaired Student's t test, ∗∗ = p

    Article Snippet: Flow CytometryAfter 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).

    Techniques: Infection, Quantitative RT-PCR