polyclonal anti human ace2 antibody  (R&D Systems)

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Mast cell activation triggered by SARS-CoV-2 causes inflammation in brain microvascular endothelial cells and microglia

doi: 10.3389/fcimb.2024.1358873

Figure Lengend Snippet: SARS-CoV-2 infection induces MC accumulation around cerebrovascular region in mice. BALB/c mice were infected intranasally with SARS-CoV-2 501Y.V2 strain at the dose of 7 × 10 4 CCID 50 . The brains were collected at 3 dpi. Brain injury was observed by H.E. staining of brain sections (A, B) and T. blue staining was used to observe MCs (E, F) . Scale bar: 50 µm. (C) ACE2 expression in hCMEC/D3 and HMC3 detected by flow cytometry with immunostaining with specific antibody. (D) Viral infection. hCMEC/D3 and HMC3 cells were infected with SARS-CoV-2 spike-pseudotyped lentivirus (HIV-NL4-3/spike) (5 ng p24 gag ) for 48h, and viral infection was determined by measuring the luciferase activity. One representative result from three independent repeats is shown. Data are presented as M ± SD . *** p < 0.001 is considered significant differences.

Article Snippet: For detecting the expression of HLA-DR and ACE2, cells were incubated with anti-human HLA-DR allophycocyanin (APC) (Tonbo Biosciences, San Diego, USA, 20-9952) or anti-human ACE2-phycoerythrin (PE) (Bioss, Beijing, China, bs-1004R) at 4°C for 30 min and detected with flow cytometry (BD Accuri C6).

Techniques: Infection, Staining, Expressing, Flow Cytometry, Immunostaining, Luciferase, Activity Assay

Journal: PLOS Pathogens

Article Title: Cellular sialoglycans are differentially required for endosomal and cell-surface entry of SARS-CoV-2 in lung cell lines

doi: 10.1371/journal.ppat.1012365

Figure Lengend Snippet: (A-B) A549 cells were transduced to stably overexpress ACE2 (A549-A), DPP4 (A549-D) in the absence or presence of TMPRSS2 (A549-AT and A549-DT). ACE2 and DPP4 expression was confirmed by western blot (A) , while TMPRSS2 expression was confirmed using flow cytometry (B) . (C-D) A549-derived or Calu-3 cells were inoculated with lentiviral particles pseudotyped with the spike proteins of MERS-CoV (C) , VSV, SARS-CoV-1, WIV1-CoV, WIV16-CoV, SARS-CoV-2 Hu1, SARS-CoV-2 delta or SARS-CoV-2 omicron (D) or pseudoparticles lacking envelope protein (no env) (C-D) for 2 h, then incubated for an additional 72 h, at which point luciferase activity was measured to assess pseudoparticle entry. The data are expressed as fold change relative to the luciferase signal obtained with no envelope. Graphs show mean +/- SEM from three independent experiments performed in triplicate.

Article Snippet: For the detection of cell-surface ACE2 expression, washed cells were resuspended in 100 μL of FACS buffer with 20 μg/mL (1:50) of anti-human ACE2 polyclonal antibody (Invitrogen #PA5-116467) for 1 h at room temperature.

Techniques: Stable Transfection, Expressing, Western Blot, Flow Cytometry, Derivative Assay, Incubation, Luciferase, Activity Assay

Journal: PLOS Pathogens

Article Title: Cellular sialoglycans are differentially required for endosomal and cell-surface entry of SARS-CoV-2 in lung cell lines

doi: 10.1371/journal.ppat.1012365

Figure Lengend Snippet: BEAS-2B cells were transduced to stably overexpress ACE2 (BEAS-2B-ACE2). Expression of ACE2 and endogenous TMPRSS2 was confirmed by western blot (A). (B) BEAS-2B-ACE2 cells were pre-treated for 1 h at 37°C with DMSO, camostat (25 μM) or E64d (10 μM) diluted in media to the indicated concentrations, then infected with SARS-CoV-2pp for 2 h at 37°C. Inocula were removed and cells were incubated in complete media for 72 h, at which point luciferase activity was measured to assess viral entry. (C-D) BEAS-2B-ACE2 cells were pre-treated with NanH diluted to 50 μg/mL in serum-free media for 30 minutes at 37°C. Cells were then washed and processed for fluorescence microscopy (C) or inoculated with SARS-CoV-2 pseudoparticles (D) . NanH-treated cells were stained with SNA-FITC (binds sialic acid) or ECL-FITC (binds galactose) diluted to final concentration of 20 μg/mL in PBS, then washed with PBS and imaged by fluorescence microscopy (10X magnification; scale bar, 200 μm). Lectin staining confirmed removal of sialic acid by the NanH treatment. (E-F) BEAS-2B-ACE2 cells were treated with protease inhibitors or NanH as described (B-C) , then infected with replication-competent recombinant VSV-SARS-CoV-2-S expressing GFP for 2 h. After 7.5 h, cells were fixed and GFP fluorescence was assessed. Representative images are shown (20X magnification; scale bar, 50 μm). The percentage of infected cells in each condition was determined using ImageJ. Graphs show mean +/- SEM from three independent experiments performed in triplicate. Statistical significance was assessed by one-way or two-way ANOVA (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; ns, not significant).

Article Snippet: For the detection of cell-surface ACE2 expression, washed cells were resuspended in 100 μL of FACS buffer with 20 μg/mL (1:50) of anti-human ACE2 polyclonal antibody (Invitrogen #PA5-116467) for 1 h at room temperature.

Techniques: Stable Transfection, Expressing, Western Blot, Infection, Incubation, Luciferase, Activity Assay, Fluorescence, Microscopy, Staining, Concentration Assay, Recombinant

Journal: Viruses

Article Title: SARS-CoV-2 Modulation of HIV Latency Reversal in a Myeloid Cell Line: Direct and Bystander Effects

doi: 10.3390/v16081310

Figure Lengend Snippet: ( A ) Relative ACE2 expression levels in different U1 and U937 cells ( left ) and representative histograms of median fluorescence intensity (MFI) for each condition ( right ). Among U1, different conditions were evaluated, such as US: unstimulated U1; cfv: U1 exposed to SARS-CoV-2 cell-free; U937: unstimulated U937. U1 exposed to conditioned media (CM) obtained from non-infected MDM (Ni), MDM exposed to SARS-CoV-2 (Wh), MDM polarized (M1, or M2). PMA (Phorbol 12-myristate 13-acetate): positive control for reversal on both HIV-latently infected cell models. Data show the mean ± SEM, and statistical significance was calculated by one-way ANOVA. ( B ) Kinetics of the SARS-CoV-2 (Wh variant, MOI: 0.1) replication in myeloid (U1, U937) cells measured using RT-qPCR targeted to N and ORF1a genes in culture supernatant, as described in M&M. T0: supernatant obtained after three cell washes. 72 h: 3 days post-infection. Data are expressed as mean ± SD obtained from 4 independent experiments. (* p < 0.05; *** p < 0.001).

Article Snippet: A rabbit anti-human ACE2 primary polyclonal Ab (ab272690, Abcam, Cambridge, UK) and goat anti-rabbit IgG secondary (PE) (Abcam, UK) were used for ACE2 quantification.

Techniques: Expressing, Fluorescence, Infection, Positive Control, Variant Assay, Quantitative RT-PCR

Journal: Viruses

Article Title: Potential Role of APOBEC3 Family Proteins in SARS-CoV-2 Replication

doi: 10.3390/v16071141

Figure Lengend Snippet: Validation of expression levels for ACE2 protein and A3 mRNAs in THP-1 parent and A3A -to- A3G -null THP-1 cells. ( A ) ACE2 protein expression levels on the surface of THP-1 parent and THP-1#11-4 ( A3A -to- A3G -null THP-1) cells. The ACE2 gene was introduced by a retroviral vector, and the expression levels of the surface ACE2 protein were detected by an anti-ACE2 polyclonal antibody (red). The number in each graph shows the percentage of ACE2 + cells compared to those stained by isotype control (gray). ( B ) A3 mRNA expression levels in Calu-3 (gray), THP-1-ACE2 (blue), and THP-1-ACE2#11-4 (red) cells. A3 mRNA expression levels were quantified by RT-qPCR and normalized to TBP mRNA levels. Each bar represents the average of three independent experiments with Standard deviation (SD). Statistical significance was determined using the two-sided unpaired t -test. *, p < 0.05 compared to THP-1 parent cells.

Article Snippet: A goat anti-ACE2 polyclonal antibody (R&D Systems, Minneapolis, MN, USA, Cat# AF933, 1:50) and an APC-conjugated donkey anti-goat IgG (R&D Systems, Cat# F0108, 1:50) were used for surface ACE2 staining ( A).

Techniques: Biomarker Discovery, Expressing, Retroviral, Plasmid Preparation, Staining, Control, Quantitative RT-PCR, Standard Deviation

Journal: Viruses

Article Title: Potential Role of APOBEC3 Family Proteins in SARS-CoV-2 Replication

doi: 10.3390/v16071141

Figure Lengend Snippet: SARS-CoV-2 replication in THP-1 parent and A3A -to- A3G -null THP-1 cells. ( A ) Replication kinetics of the Wuhan, BA.1, and BA.5 variants produced from THP-1 parent and THP-1#11-4 ( A3A -to- A3G -null THP-1) cells without (blue line) or with (red line) ACE2 protein expression. The SARS-CoV-2 N gene was quantified by RT-qPCR to monitor the viral RNA copy number across the indicated time points. Each timepoint represents the average of four independent experiments with SD. ( B ) Passage experiments. The SARS-CoV-2 N gene in the cell culture supernatants produced from THP-1-ACE2 or THP-1-ACE2#11-4 ( A3A -to- A3G -null THP-1) cells at 96 h postinfection of each passage were quantified by RT-qPCR to monitor the viral RNA copy number of the Wuhan (gray), BA.1 (blue), and BA.5 (red) variants. Each bar represents the average of three independent experiments with SD.

Article Snippet: A goat anti-ACE2 polyclonal antibody (R&D Systems, Minneapolis, MN, USA, Cat# AF933, 1:50) and an APC-conjugated donkey anti-goat IgG (R&D Systems, Cat# F0108, 1:50) were used for surface ACE2 staining ( A).

Techniques: Produced, Expressing, Quantitative RT-PCR, Cell Culture

Journal: Viruses

Article Title: Potential Role of APOBEC3 Family Proteins in SARS-CoV-2 Replication

doi: 10.3390/v16071141

Figure Lengend Snippet: SARS-CoV-2 infectivity produced from THP-1 parent and A3A -to- A3G -null THP-1 cells during passage experiments. ( A ) Representative pictures of plaque assay. Cell culture supernatants obtained from the passage experiments for the Wuhan variant were also used for plaque assay with serial 10-times dilution. ( B ) PFU/mL of the Wuhan variant produced from THP-1-ACE2 (gray) or THP-1-ACE2#11-4 ( A3A -to- A3G -null THP-1) (blue) cells at 96 h postinfection during passage experiments.

Article Snippet: A goat anti-ACE2 polyclonal antibody (R&D Systems, Minneapolis, MN, USA, Cat# AF933, 1:50) and an APC-conjugated donkey anti-goat IgG (R&D Systems, Cat# F0108, 1:50) were used for surface ACE2 staining ( A).

Techniques: Infection, Produced, Plaque Assay, Cell Culture, Variant Assay

Journal: Viruses

Article Title: Potential Role of APOBEC3 Family Proteins in SARS-CoV-2 Replication

doi: 10.3390/v16071141

Figure Lengend Snippet: Analysis of mutations in SARS-CoV-2 genomes produced from THP-1 parent and A3A -to- A3G -null THP-1 cells during passage experiments. SARS-CoV-2 genomic RNA was isolated and subjected to WGS. Sample 1 to 4: Wuhan variant from passage 1 of THP-1-ACE2 cells. Sample 5 to 8: Wuhan variant from passage 5 of THP-1-ACE2 cells. Sample 9 to 12: Wuhan variant from passage 1 of THP-1-ACE2#11-4 ( A3A -to- A3G -null THP-1) cells. Sample 13 and 14: Wuhan variant from passage 3 of THP-1-ACE2#11-4 cells. Green boxes on the top show each SARS-CoV-2 ORF gene with nucleotide position.

Article Snippet: A goat anti-ACE2 polyclonal antibody (R&D Systems, Minneapolis, MN, USA, Cat# AF933, 1:50) and an APC-conjugated donkey anti-goat IgG (R&D Systems, Cat# F0108, 1:50) were used for surface ACE2 staining ( A).

Techniques: Produced, Isolation, Variant Assay