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anti clec5a mab  (R&D Systems)


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    R&D Systems anti clec5a mab
    (a&b) Neutrophils (4 × 10 5 /ml) from healthy donors were incubated with SARS-CoV-2 (MOI = 1) with or without autologous platelets (4 × 10 6 /ml) for 5 h at 37 °C. The scale bar is 10 μm. The detailed structure of SARS-CoV-2-induced NET formation was observed under a confocal microscope (Leica). NET formation was visualized by fluorescent staining of DNA (blue), histone (green), and MPO (red) (a) . NETs level was measured by MetaMorph software and presented as Cit-H3 area (mm 2 ) (b) . (c) Human neutrophils (4 × 10 5 /ml) were pretreated with anti-hCLEC5A mAb (3E12A2, 100 μg/ml), anti-TLR2 mAb (# MAB2616, 100 μg/ml), or combination of both antibodies for 30 min at room temperature, followed by incubation with SARS-CoV-2 (MOI = 0.1 and 1) in the presence or absence of platelets (4 × 10 6 /ml) for 5 h and 20 h. The level of NET formation was determined by histone area (μm 2 ). (d) Neutrophils (4 × 10 5 /ml) from WT, <t>clec5a</t> -/- tlr2 -/- , and clec5a -/- tlr2 -/- mice were incubated with SARS-CoV-2 (MOI = 1) in the presence or absence of WT platelets (4 × 10 6 /ml) for 5 h at 37 °C. (e) Human neutrophils were pre-treated with anti-hCLEC5A mAb (3E12A2, 100 μg/ml), anti-TLR2 mAb (# MAB2616, 100 μg/ml), or combination of both antibodies for 30 min at room temperature, followed by incubation with SARS-CoV-2 spike pseudotyped virus (MOI = 0.1) in the presence or absence of autologous platelets (4 × 10 6 /ml) for 3 h. Data are mean ± SEM and repeats of 3 to 5 independent experiments. *p<0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (Student’s t-test).
    Anti Clec5a Mab, supplied by R&D Systems, used in various techniques. Bioz Stars score: 94/100, based on 125 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/anti+clec5a+mab/Human+CD3+epsilon+Antibody/bio_rxiv__2022__02__01__478701-68-10-20
    Average 94 stars, based on 125 article reviews
    anti clec5a mab - by Bioz Stars, 2026-10
    94/100 stars

    Images

    1) Product Images from "CLEC5A and TLR2 are critical in SARS-CoV-2-induced NET formation and lung inflammation"

    Article Title: CLEC5A and TLR2 are critical in SARS-CoV-2-induced NET formation and lung inflammation

    Journal: bioRxiv

    doi: 10.1101/2022.02.01.478701

    (a&b) Neutrophils (4 × 10 5 /ml) from healthy donors were incubated with SARS-CoV-2 (MOI = 1) with or without autologous platelets (4 × 10 6 /ml) for 5 h at 37 °C. The scale bar is 10 μm. The detailed structure of SARS-CoV-2-induced NET formation was observed under a confocal microscope (Leica). NET formation was visualized by fluorescent staining of DNA (blue), histone (green), and MPO (red) (a) . NETs level was measured by MetaMorph software and presented as Cit-H3 area (mm 2 ) (b) . (c) Human neutrophils (4 × 10 5 /ml) were pretreated with anti-hCLEC5A mAb (3E12A2, 100 μg/ml), anti-TLR2 mAb (# MAB2616, 100 μg/ml), or combination of both antibodies for 30 min at room temperature, followed by incubation with SARS-CoV-2 (MOI = 0.1 and 1) in the presence or absence of platelets (4 × 10 6 /ml) for 5 h and 20 h. The level of NET formation was determined by histone area (μm 2 ). (d) Neutrophils (4 × 10 5 /ml) from WT, clec5a -/- tlr2 -/- , and clec5a -/- tlr2 -/- mice were incubated with SARS-CoV-2 (MOI = 1) in the presence or absence of WT platelets (4 × 10 6 /ml) for 5 h at 37 °C. (e) Human neutrophils were pre-treated with anti-hCLEC5A mAb (3E12A2, 100 μg/ml), anti-TLR2 mAb (# MAB2616, 100 μg/ml), or combination of both antibodies for 30 min at room temperature, followed by incubation with SARS-CoV-2 spike pseudotyped virus (MOI = 0.1) in the presence or absence of autologous platelets (4 × 10 6 /ml) for 3 h. Data are mean ± SEM and repeats of 3 to 5 independent experiments. *p<0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (Student’s t-test).
    Figure Legend Snippet: (a&b) Neutrophils (4 × 10 5 /ml) from healthy donors were incubated with SARS-CoV-2 (MOI = 1) with or without autologous platelets (4 × 10 6 /ml) for 5 h at 37 °C. The scale bar is 10 μm. The detailed structure of SARS-CoV-2-induced NET formation was observed under a confocal microscope (Leica). NET formation was visualized by fluorescent staining of DNA (blue), histone (green), and MPO (red) (a) . NETs level was measured by MetaMorph software and presented as Cit-H3 area (mm 2 ) (b) . (c) Human neutrophils (4 × 10 5 /ml) were pretreated with anti-hCLEC5A mAb (3E12A2, 100 μg/ml), anti-TLR2 mAb (# MAB2616, 100 μg/ml), or combination of both antibodies for 30 min at room temperature, followed by incubation with SARS-CoV-2 (MOI = 0.1 and 1) in the presence or absence of platelets (4 × 10 6 /ml) for 5 h and 20 h. The level of NET formation was determined by histone area (μm 2 ). (d) Neutrophils (4 × 10 5 /ml) from WT, clec5a -/- tlr2 -/- , and clec5a -/- tlr2 -/- mice were incubated with SARS-CoV-2 (MOI = 1) in the presence or absence of WT platelets (4 × 10 6 /ml) for 5 h at 37 °C. (e) Human neutrophils were pre-treated with anti-hCLEC5A mAb (3E12A2, 100 μg/ml), anti-TLR2 mAb (# MAB2616, 100 μg/ml), or combination of both antibodies for 30 min at room temperature, followed by incubation with SARS-CoV-2 spike pseudotyped virus (MOI = 0.1) in the presence or absence of autologous platelets (4 × 10 6 /ml) for 3 h. Data are mean ± SEM and repeats of 3 to 5 independent experiments. *p<0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (Student’s t-test).

    Techniques Used: Incubation, Microscopy, Staining, Software, Virus

    (a) EVs from healthy controls (HCs-EVs, n=5) and COVID-19 patients (COVID19-EVs, n=5) were harvested by ultracentrifugation, then lysed in RIPA solution before subjected to mass spectrometry analysis. Proteins expressed in COVID-19 EVs, but not in HCs EVs, were further analyzed using the QIAGEN Ingenuity Pathway Analysis (QIAGEN IPA) software. Proteins which were expressed in all the COVID19-EVs were displayed. (b&c) HCs-EVs (n=10) and COVID19-EVs (n=10) were analyzed by flow cytometry, and markers highly activated in COVID-19 platelets were expressed as a heat map (b) or by mean fluorescence intensity (c) . (d) Neutrophils were pre-incubated with anti-CLEC5A mAb (3E12A2, 100 μg/ml), anti-TLR2 mAb (# MAB2616, 100 μg/ml), or both anti-CLEC5A mAb (3E12A2, 100 μg/ml) and anti-TLR2 mAb (# MAB2616, 100 μg/ml), for 30 min at room temperature, followed by incubation with EVs (1 μg/ml) from COVID-19 patients (n=6) at 37°C for 3 h. Data are mean ± sd and repeats of at least three independent experiments. * p <0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (Student’s t -test).
    Figure Legend Snippet: (a) EVs from healthy controls (HCs-EVs, n=5) and COVID-19 patients (COVID19-EVs, n=5) were harvested by ultracentrifugation, then lysed in RIPA solution before subjected to mass spectrometry analysis. Proteins expressed in COVID-19 EVs, but not in HCs EVs, were further analyzed using the QIAGEN Ingenuity Pathway Analysis (QIAGEN IPA) software. Proteins which were expressed in all the COVID19-EVs were displayed. (b&c) HCs-EVs (n=10) and COVID19-EVs (n=10) were analyzed by flow cytometry, and markers highly activated in COVID-19 platelets were expressed as a heat map (b) or by mean fluorescence intensity (c) . (d) Neutrophils were pre-incubated with anti-CLEC5A mAb (3E12A2, 100 μg/ml), anti-TLR2 mAb (# MAB2616, 100 μg/ml), or both anti-CLEC5A mAb (3E12A2, 100 μg/ml) and anti-TLR2 mAb (# MAB2616, 100 μg/ml), for 30 min at room temperature, followed by incubation with EVs (1 μg/ml) from COVID-19 patients (n=6) at 37°C for 3 h. Data are mean ± sd and repeats of at least three independent experiments. * p <0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (Student’s t -test).

    Techniques Used: Mass Spectrometry, Software, Flow Cytometry, Fluorescence, Incubation

    C57BL/6 mice (WT) (n=3) and clec5a -/- tlr2 -/- mice (n=3)were inoculated with AAV-hACE2 for 14 days, followed by intranasal inoculation of SARS-CoV-2 (8 × 10 4 PFU/per mice). Tissues were collected at 3 days and 5 days post-infection. (a) The level of proinflammatory cytokines and chemokines were measured by real-time PCR and presented as fold change (compared to AAV-hACE2 uninfected mice/mock). ( b-d) NET structure and thrombus were detected by Hoechst. 33342 (blue), anti-MPO antibody (green), anti-citrullinated histone H3 (red), anti-CD42b antibody (yellow) (b) , and images were captured by a confocal microscope and subjected to determine the area of MPO (c) and CD42b (d) using MetaMorph TM software. (e) Cell infiltrated to lung. Interstitial macrophage (interstitial MΦ) was defined as CD11b + CD64 + F4/80 + cells; monocyte-derived dendritic cell (DC)/macrophage (MΦ) was defined as CD11b + CD64 + Ly6C + ; Ly6C + monocyte was defined as Ly6C + . The cell number of each cell population was calculated using the multiple fluorescent staining image and analyzed by software MetaMorph TM , and the data was presented as cell number/ per 664225 (815 × 815). Scale bar is 200 μm. Data are represented as mean ± SEM. * p <0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 (Student’s t -test).
    Figure Legend Snippet: C57BL/6 mice (WT) (n=3) and clec5a -/- tlr2 -/- mice (n=3)were inoculated with AAV-hACE2 for 14 days, followed by intranasal inoculation of SARS-CoV-2 (8 × 10 4 PFU/per mice). Tissues were collected at 3 days and 5 days post-infection. (a) The level of proinflammatory cytokines and chemokines were measured by real-time PCR and presented as fold change (compared to AAV-hACE2 uninfected mice/mock). ( b-d) NET structure and thrombus were detected by Hoechst. 33342 (blue), anti-MPO antibody (green), anti-citrullinated histone H3 (red), anti-CD42b antibody (yellow) (b) , and images were captured by a confocal microscope and subjected to determine the area of MPO (c) and CD42b (d) using MetaMorph TM software. (e) Cell infiltrated to lung. Interstitial macrophage (interstitial MΦ) was defined as CD11b + CD64 + F4/80 + cells; monocyte-derived dendritic cell (DC)/macrophage (MΦ) was defined as CD11b + CD64 + Ly6C + ; Ly6C + monocyte was defined as Ly6C + . The cell number of each cell population was calculated using the multiple fluorescent staining image and analyzed by software MetaMorph TM , and the data was presented as cell number/ per 664225 (815 × 815). Scale bar is 200 μm. Data are represented as mean ± SEM. * p <0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 (Student’s t -test).

    Techniques Used: Infection, Real-time Polymerase Chain Reaction, Microscopy, Software, Derivative Assay, Staining

    Related Articles

    Blocking Assay:

    Article Title: CLEC5A and TLR2 are critical in SARS-CoV-2-induced NET formation and lung inflammation
    Article Snippet: .. For blocking assay, neutrophils were preincubated with isotype (100 μg/ml), anti-CLEC5A mAb (100 μg/ml, clone 3E12A2), anti-TLR2 mAb (100 μg/ml, R&D system), or a mixture of anti-CLEC5A mAb and anti-TLR2 mAb for 30 min at room temperature before incubation with SARS-CoV-2. ..

    Incubation:

    Article Title: CLEC5A and TLR2 are critical in SARS-CoV-2-induced NET formation and lung inflammation
    Article Snippet: .. For blocking assay, neutrophils were preincubated with isotype (100 μg/ml), anti-CLEC5A mAb (100 μg/ml, clone 3E12A2), anti-TLR2 mAb (100 μg/ml, R&D system), or a mixture of anti-CLEC5A mAb and anti-TLR2 mAb for 30 min at room temperature before incubation with SARS-CoV-2. ..

    Expressing:

    Article Title: CLEC5A is critical for dengue-virus-induced lethal disease.
    Article Snippet: Fusion proteins (1mg per well) were coated on microtitre plates to capture DV2 (5 3 106 plaque-forming units per well), and the bound dengue virions were detected by biotinylated anti-DV envelope protein antibody26 and HRP-conjugated streptavidin with 3,39,5,59- tetramethylbenzidine (TMB) (BD Pharmingen) substrate. .. To characterize the expression pattern of CLEC5A and DC-SIGN, cells were stained with fluorescein isothiocyanate (FITC)-conjugated anti-CLEC5A mAb (clone 283834; R&D Systems) or FITC-conjugated anti-DC-SIGN mAb (BD Pharmingen) in conjunction with phycoerythrin (PE)-conjugated mAbs against CD3, CD19, CD56, CD14 or CD66 (BD Pharmingen). .. Results were compared with those obtained using isotype-matched controls (IgG2b for anti-CLEC5A mAb, IgG1 for antiDC-SIGN; Sigma).

    Staining:

    Article Title: CLEC5A is critical for dengue-virus-induced lethal disease.
    Article Snippet: Fusion proteins (1mg per well) were coated on microtitre plates to capture DV2 (5 3 106 plaque-forming units per well), and the bound dengue virions were detected by biotinylated anti-DV envelope protein antibody26 and HRP-conjugated streptavidin with 3,39,5,59- tetramethylbenzidine (TMB) (BD Pharmingen) substrate. .. To characterize the expression pattern of CLEC5A and DC-SIGN, cells were stained with fluorescein isothiocyanate (FITC)-conjugated anti-CLEC5A mAb (clone 283834; R&D Systems) or FITC-conjugated anti-DC-SIGN mAb (BD Pharmingen) in conjunction with phycoerythrin (PE)-conjugated mAbs against CD3, CD19, CD56, CD14 or CD66 (BD Pharmingen). .. Results were compared with those obtained using isotype-matched controls (IgG2b for anti-CLEC5A mAb, IgG1 for antiDC-SIGN; Sigma).



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    (a&b) Neutrophils (4 × 10 5 /ml) from healthy donors were incubated with SARS-CoV-2 (MOI = 1) with or without autologous platelets (4 × 10 6 /ml) for 5 h at 37 °C. The scale bar is 10 μm. The detailed structure of SARS-CoV-2-induced NET formation was observed under a confocal microscope (Leica). NET formation was visualized by fluorescent staining of DNA (blue), histone (green), and MPO (red) (a) . NETs level was measured by MetaMorph software and presented as Cit-H3 area (mm 2 ) (b) . (c) Human neutrophils (4 × 10 5 /ml) were pretreated with anti-hCLEC5A mAb (3E12A2, 100 μg/ml), anti-TLR2 mAb (# MAB2616, 100 μg/ml), or combination of both antibodies for 30 min at room temperature, followed by incubation with SARS-CoV-2 (MOI = 0.1 and 1) in the presence or absence of platelets (4 × 10 6 /ml) for 5 h and 20 h. The level of NET formation was determined by histone area (μm 2 ). (d) Neutrophils (4 × 10 5 /ml) from WT, <t>clec5a</t> -/- tlr2 -/- , and clec5a -/- tlr2 -/- mice were incubated with SARS-CoV-2 (MOI = 1) in the presence or absence of WT platelets (4 × 10 6 /ml) for 5 h at 37 °C. (e) Human neutrophils were pre-treated with anti-hCLEC5A mAb (3E12A2, 100 μg/ml), anti-TLR2 mAb (# MAB2616, 100 μg/ml), or combination of both antibodies for 30 min at room temperature, followed by incubation with SARS-CoV-2 spike pseudotyped virus (MOI = 0.1) in the presence or absence of autologous platelets (4 × 10 6 /ml) for 3 h. Data are mean ± SEM and repeats of 3 to 5 independent experiments. *p<0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (Student’s t-test).
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    (a&b) Neutrophils (4 × 10 5 /ml) from healthy donors were incubated with SARS-CoV-2 (MOI = 1) with or without autologous platelets (4 × 10 6 /ml) for 5 h at 37 °C. The scale bar is 10 μm. The detailed structure of SARS-CoV-2-induced NET formation was observed under a confocal microscope (Leica). NET formation was visualized by fluorescent staining of DNA (blue), histone (green), and MPO (red) (a) . NETs level was measured by MetaMorph software and presented as Cit-H3 area (mm 2 ) (b) . (c) Human neutrophils (4 × 10 5 /ml) were pretreated with anti-hCLEC5A mAb (3E12A2, 100 μg/ml), anti-TLR2 mAb (# MAB2616, 100 μg/ml), or combination of both antibodies for 30 min at room temperature, followed by incubation with SARS-CoV-2 (MOI = 0.1 and 1) in the presence or absence of platelets (4 × 10 6 /ml) for 5 h and 20 h. The level of NET formation was determined by histone area (μm 2 ). (d) Neutrophils (4 × 10 5 /ml) from WT, clec5a -/- tlr2 -/- , and clec5a -/- tlr2 -/- mice were incubated with SARS-CoV-2 (MOI = 1) in the presence or absence of WT platelets (4 × 10 6 /ml) for 5 h at 37 °C. (e) Human neutrophils were pre-treated with anti-hCLEC5A mAb (3E12A2, 100 μg/ml), anti-TLR2 mAb (# MAB2616, 100 μg/ml), or combination of both antibodies for 30 min at room temperature, followed by incubation with SARS-CoV-2 spike pseudotyped virus (MOI = 0.1) in the presence or absence of autologous platelets (4 × 10 6 /ml) for 3 h. Data are mean ± SEM and repeats of 3 to 5 independent experiments. *p<0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (Student’s t-test).

    Journal: bioRxiv

    Article Title: CLEC5A and TLR2 are critical in SARS-CoV-2-induced NET formation and lung inflammation

    doi: 10.1101/2022.02.01.478701

    Figure Lengend Snippet: (a&b) Neutrophils (4 × 10 5 /ml) from healthy donors were incubated with SARS-CoV-2 (MOI = 1) with or without autologous platelets (4 × 10 6 /ml) for 5 h at 37 °C. The scale bar is 10 μm. The detailed structure of SARS-CoV-2-induced NET formation was observed under a confocal microscope (Leica). NET formation was visualized by fluorescent staining of DNA (blue), histone (green), and MPO (red) (a) . NETs level was measured by MetaMorph software and presented as Cit-H3 area (mm 2 ) (b) . (c) Human neutrophils (4 × 10 5 /ml) were pretreated with anti-hCLEC5A mAb (3E12A2, 100 μg/ml), anti-TLR2 mAb (# MAB2616, 100 μg/ml), or combination of both antibodies for 30 min at room temperature, followed by incubation with SARS-CoV-2 (MOI = 0.1 and 1) in the presence or absence of platelets (4 × 10 6 /ml) for 5 h and 20 h. The level of NET formation was determined by histone area (μm 2 ). (d) Neutrophils (4 × 10 5 /ml) from WT, clec5a -/- tlr2 -/- , and clec5a -/- tlr2 -/- mice were incubated with SARS-CoV-2 (MOI = 1) in the presence or absence of WT platelets (4 × 10 6 /ml) for 5 h at 37 °C. (e) Human neutrophils were pre-treated with anti-hCLEC5A mAb (3E12A2, 100 μg/ml), anti-TLR2 mAb (# MAB2616, 100 μg/ml), or combination of both antibodies for 30 min at room temperature, followed by incubation with SARS-CoV-2 spike pseudotyped virus (MOI = 0.1) in the presence or absence of autologous platelets (4 × 10 6 /ml) for 3 h. Data are mean ± SEM and repeats of 3 to 5 independent experiments. *p<0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (Student’s t-test).

    Article Snippet: For blocking assay, neutrophils were preincubated with isotype (100 μg/ml), anti-CLEC5A mAb (100 μg/ml, clone 3E12A2), anti-TLR2 mAb (100 μg/ml, R&D system), or a mixture of anti-CLEC5A mAb and anti-TLR2 mAb for 30 min at room temperature before incubation with SARS-CoV-2.

    Techniques: Incubation, Microscopy, Staining, Software, Virus

    (a) EVs from healthy controls (HCs-EVs, n=5) and COVID-19 patients (COVID19-EVs, n=5) were harvested by ultracentrifugation, then lysed in RIPA solution before subjected to mass spectrometry analysis. Proteins expressed in COVID-19 EVs, but not in HCs EVs, were further analyzed using the QIAGEN Ingenuity Pathway Analysis (QIAGEN IPA) software. Proteins which were expressed in all the COVID19-EVs were displayed. (b&c) HCs-EVs (n=10) and COVID19-EVs (n=10) were analyzed by flow cytometry, and markers highly activated in COVID-19 platelets were expressed as a heat map (b) or by mean fluorescence intensity (c) . (d) Neutrophils were pre-incubated with anti-CLEC5A mAb (3E12A2, 100 μg/ml), anti-TLR2 mAb (# MAB2616, 100 μg/ml), or both anti-CLEC5A mAb (3E12A2, 100 μg/ml) and anti-TLR2 mAb (# MAB2616, 100 μg/ml), for 30 min at room temperature, followed by incubation with EVs (1 μg/ml) from COVID-19 patients (n=6) at 37°C for 3 h. Data are mean ± sd and repeats of at least three independent experiments. * p <0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (Student’s t -test).

    Journal: bioRxiv

    Article Title: CLEC5A and TLR2 are critical in SARS-CoV-2-induced NET formation and lung inflammation

    doi: 10.1101/2022.02.01.478701

    Figure Lengend Snippet: (a) EVs from healthy controls (HCs-EVs, n=5) and COVID-19 patients (COVID19-EVs, n=5) were harvested by ultracentrifugation, then lysed in RIPA solution before subjected to mass spectrometry analysis. Proteins expressed in COVID-19 EVs, but not in HCs EVs, were further analyzed using the QIAGEN Ingenuity Pathway Analysis (QIAGEN IPA) software. Proteins which were expressed in all the COVID19-EVs were displayed. (b&c) HCs-EVs (n=10) and COVID19-EVs (n=10) were analyzed by flow cytometry, and markers highly activated in COVID-19 platelets were expressed as a heat map (b) or by mean fluorescence intensity (c) . (d) Neutrophils were pre-incubated with anti-CLEC5A mAb (3E12A2, 100 μg/ml), anti-TLR2 mAb (# MAB2616, 100 μg/ml), or both anti-CLEC5A mAb (3E12A2, 100 μg/ml) and anti-TLR2 mAb (# MAB2616, 100 μg/ml), for 30 min at room temperature, followed by incubation with EVs (1 μg/ml) from COVID-19 patients (n=6) at 37°C for 3 h. Data are mean ± sd and repeats of at least three independent experiments. * p <0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (Student’s t -test).

    Article Snippet: For blocking assay, neutrophils were preincubated with isotype (100 μg/ml), anti-CLEC5A mAb (100 μg/ml, clone 3E12A2), anti-TLR2 mAb (100 μg/ml, R&D system), or a mixture of anti-CLEC5A mAb and anti-TLR2 mAb for 30 min at room temperature before incubation with SARS-CoV-2.

    Techniques: Mass Spectrometry, Software, Flow Cytometry, Fluorescence, Incubation

    C57BL/6 mice (WT) (n=3) and clec5a -/- tlr2 -/- mice (n=3)were inoculated with AAV-hACE2 for 14 days, followed by intranasal inoculation of SARS-CoV-2 (8 × 10 4 PFU/per mice). Tissues were collected at 3 days and 5 days post-infection. (a) The level of proinflammatory cytokines and chemokines were measured by real-time PCR and presented as fold change (compared to AAV-hACE2 uninfected mice/mock). ( b-d) NET structure and thrombus were detected by Hoechst. 33342 (blue), anti-MPO antibody (green), anti-citrullinated histone H3 (red), anti-CD42b antibody (yellow) (b) , and images were captured by a confocal microscope and subjected to determine the area of MPO (c) and CD42b (d) using MetaMorph TM software. (e) Cell infiltrated to lung. Interstitial macrophage (interstitial MΦ) was defined as CD11b + CD64 + F4/80 + cells; monocyte-derived dendritic cell (DC)/macrophage (MΦ) was defined as CD11b + CD64 + Ly6C + ; Ly6C + monocyte was defined as Ly6C + . The cell number of each cell population was calculated using the multiple fluorescent staining image and analyzed by software MetaMorph TM , and the data was presented as cell number/ per 664225 (815 × 815). Scale bar is 200 μm. Data are represented as mean ± SEM. * p <0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 (Student’s t -test).

    Journal: bioRxiv

    Article Title: CLEC5A and TLR2 are critical in SARS-CoV-2-induced NET formation and lung inflammation

    doi: 10.1101/2022.02.01.478701

    Figure Lengend Snippet: C57BL/6 mice (WT) (n=3) and clec5a -/- tlr2 -/- mice (n=3)were inoculated with AAV-hACE2 for 14 days, followed by intranasal inoculation of SARS-CoV-2 (8 × 10 4 PFU/per mice). Tissues were collected at 3 days and 5 days post-infection. (a) The level of proinflammatory cytokines and chemokines were measured by real-time PCR and presented as fold change (compared to AAV-hACE2 uninfected mice/mock). ( b-d) NET structure and thrombus were detected by Hoechst. 33342 (blue), anti-MPO antibody (green), anti-citrullinated histone H3 (red), anti-CD42b antibody (yellow) (b) , and images were captured by a confocal microscope and subjected to determine the area of MPO (c) and CD42b (d) using MetaMorph TM software. (e) Cell infiltrated to lung. Interstitial macrophage (interstitial MΦ) was defined as CD11b + CD64 + F4/80 + cells; monocyte-derived dendritic cell (DC)/macrophage (MΦ) was defined as CD11b + CD64 + Ly6C + ; Ly6C + monocyte was defined as Ly6C + . The cell number of each cell population was calculated using the multiple fluorescent staining image and analyzed by software MetaMorph TM , and the data was presented as cell number/ per 664225 (815 × 815). Scale bar is 200 μm. Data are represented as mean ± SEM. * p <0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 (Student’s t -test).

    Article Snippet: For blocking assay, neutrophils were preincubated with isotype (100 μg/ml), anti-CLEC5A mAb (100 μg/ml, clone 3E12A2), anti-TLR2 mAb (100 μg/ml, R&D system), or a mixture of anti-CLEC5A mAb and anti-TLR2 mAb for 30 min at room temperature before incubation with SARS-CoV-2.

    Techniques: Infection, Real-time Polymerase Chain Reaction, Microscopy, Software, Derivative Assay, Staining