Journal: Biosensors & Bioelectronics

Article Title: Smartphone-based sensitive detection of SARS-CoV-2 from saline gargle samples via flow profile analysis on a paper microfluidic chip

doi: 10.1016/j.bios.2022.114192

Figure Lengend Snippet: Flow profile assay of SARS-CoV-2 on a paper-based microfluidic chip. (A) Paper-based microfluidic chip design containing green edge and three red squares for recognizing the chip area in automated flow distance measurement. (B) The chip holder and the chip lock. (C) A paper-based microfluidic chip was placed into a chip holder to flatten the chip. (D) 4 μL of sample was loaded directly onto the square inlet (top) of each channel and dried for 10 min. (E) A smartphone camera was held just above the chip to view the chip area and start recording the video. (F) 4 μL of Ab-particles were loaded, and the liquid flow on the paper microfluidic chip was recorded with the smartphone camera.

Article Snippet: Two types of antibodies were used in this study: rabbit monoclonal antibody to SARS-CoV-2 (2019-nCoV) nucleocapsid (Sino Biological US Inc., PA, USA) and rabbit polyclonal antibody to SARS-CoV/SARS-CoV-2 nucleocapsid (Sino Biological US Inc., PA, USA).

Techniques:

Journal: Biosensors & Bioelectronics

Article Title: Smartphone-based sensitive detection of SARS-CoV-2 from saline gargle samples via flow profile analysis on a paper microfluidic chip

doi: 10.1016/j.bios.2022.114192

Figure Lengend Snippet: Video processing algorithm. Python code was developed to automatically obtain the flow distance over time. (A) The red squares were detected, and each frame is rotated for orientation correction. (B) The green edge was recognized and cropped. (C) The cropped area was analyzed to generate an intensity histogram plot. Using appropriate thresholding, the liquid flow was recognized on paper. (D) The flow on each channel was read separately by recognizing it as the black pixels increasing along the vertical centerline. (E) Flow distance vs. time profile of mAb-particles on the preloaded 1000 fg/μL SARS-CoV-2 spiked sample. (F) Flow distance vs. time profile of pAb-particles on the preloaded 1000 fg/μL SARS-CoV-2 spiked sample.

Article Snippet: Two types of antibodies were used in this study: rabbit monoclonal antibody to SARS-CoV-2 (2019-nCoV) nucleocapsid (Sino Biological US Inc., PA, USA) and rabbit polyclonal antibody to SARS-CoV/SARS-CoV-2 nucleocapsid (Sino Biological US Inc., PA, USA).

Techniques:

Journal: Biosensors & Bioelectronics

Article Title: Smartphone-based sensitive detection of SARS-CoV-2 from saline gargle samples via flow profile analysis on a paper microfluidic chip

doi: 10.1016/j.bios.2022.114192

Figure Lengend Snippet: Assay LOD and Specificity. NC indicates negative control and * shows p < 0.05 between sample and NC using one-tailed student's t-test with unequal variance. Error bars represent standard error. (A) Flow distances at 30 s on the paper microfluidic chips preloaded with SARS-CoV-2 spiked in 1% v/v human pooled saliva, using polyclonal antibody conjugated particles at 0.04 μg/μL (n = 3). (B) Flow distances at 30 s on the paper microfluidic chips preloaded with SARS-CoV-2 spiked in simulated saline gargle samples (∼15% v/v saliva and 0.9% saline), using polyclonal antibody conjugated particles at 0.04 μg/μL, with the addition of 0.5% w/v Tween 20 (n = 3). (C) Specificity test results with 1 pg/μL SARS-CoV-2 and influenza A/H1N1 (Ct values of 25–28) spiked in 1% v/v and 10% v/v saliva in 0.9% saline using the pAb-particles, shown together with the no target control samples (1% or 10% saliva in 0.9% saline) (n = 3).

Article Snippet: Two types of antibodies were used in this study: rabbit monoclonal antibody to SARS-CoV-2 (2019-nCoV) nucleocapsid (Sino Biological US Inc., PA, USA) and rabbit polyclonal antibody to SARS-CoV/SARS-CoV-2 nucleocapsid (Sino Biological US Inc., PA, USA).

Techniques: Negative Control, One-tailed Test

Journal: Biosensors & Bioelectronics

Article Title: Smartphone-based sensitive detection of SARS-CoV-2 from saline gargle samples via flow profile analysis on a paper microfluidic chip

doi: 10.1016/j.bios.2022.114192

Figure Lengend Snippet: Turbidity assessment of clinical saline gargle samples. Error bars represent standard error. (A) Photographs of negative and positive clinical saline gargle samples, obtained from human subjects. The normalized turbidity was determined by comparing the pixel intensities of the sample tubes against the black background. Red boxes indicate samples that were determined to be turbid using the procedure described in part B. (B) Using the normalized (to empty tube) turbidity, all clinical samples were classified into two categories, turbid and clear, using the threshold value of 1.41. Note: while all samples were classified in this manner, some could not undergo all subsequent testing due to low sample volume. (C) Surface tension measurements showed a decreasing trend with increased turbidity. (D) Total protein concentration of samples according to the Bradford assay. Turbid and clear samples showed no difference in total protein concentration, but SARS-CoV-2 positive samples had a higher (not significant) total protein concentration than negative samples. (n = 5 for negative clear, n = 3 for negative turbid, n = 6 for positive clear, and n = 4 for positive turbid). (E) Samples with a last oral intake (LOI) of 10–30 min prior to sample acquisition (n = 6) had higher turbidity than samples with a longer time since LOI (60+ min; n = 10), and the difference was statistically significant ( p < 0.05). Average values are shown in the bar chart. (F) The time to constant velocity (n = 2) and surface tension of no toothpaste vs. toothpaste-added (10 mg/mL) NC samples, along with photos of the samples. Surface tension was measured at 0, 2, 4, 6, 8, and 10 s and the stabilized final value was chosen (hence no error bar). The accuracy of surface tension measurement is less than 1 mN/mm.

Article Snippet: Two types of antibodies were used in this study: rabbit monoclonal antibody to SARS-CoV-2 (2019-nCoV) nucleocapsid (Sino Biological US Inc., PA, USA) and rabbit polyclonal antibody to SARS-CoV/SARS-CoV-2 nucleocapsid (Sino Biological US Inc., PA, USA).

Techniques: Protein Concentration, Bradford Assay

Journal: Cell Stem Cell

Article Title: SARS-CoV-2 infects the human kidney and drives fibrosis in kidney organoids

doi: 10.1016/j.stem.2021.12.010

Figure Lengend Snippet:

Article Snippet: anti-human SARS-CoV-2 nucleoprotein, monoclonal mouse IgG1 , Sino Biological Europe , Cat#40143-MM05; RRID: AB_2827977.

Techniques: Virus, Control, Recombinant, Membrane, Cell Culture, Sterility, Reverse Transcription, RNAscope, Multiplex Assay, Expressing, Software

Journal: Cell Reports

Article Title: A two-adjuvant multiantigen candidate vaccine induces superior protective immune responses against SARS-CoV-2 challenge

doi: 10.1016/j.celrep.2021.110112

Figure Lengend Snippet: Neutralization of SARS-CoV-2 pseudovirus in sera and detection of N- and S-specific T cell responses against the B.1351 and B.1.617.2 variants SARS-CoV-2 wild-type (WT), B.1.351 variant, or B.1.617 variant pseudoviruses were incubated with different serum sample dilutions for 1 h at 37°C before the mixtures were added to ACE2-overexpressing 293T cells. Transduction efficiency was quantified by measuring virus-encoded luciferase activity in cell lysates 48 h after transduction and used to calculate the serum dilution factor that resulted in a 50% reduction in pseudovirus particles that were associated with different degrees of S protein-mediated cell entry. (A and B) The 50% pseudovirus neutralization (pVNT 50 ) in serum from mice immunized with the candidate vaccine (A) and mice immunized with the inactivated vaccine (B) against the B.1.351 and B.1.617.2 variants compared with that against the wild-type (WT) virus (n = 4 mice per group). (C–F) Antigen-specific activation of T cells by the N and S proteins of the B.1.351 and B.1.617.2 variants compared with the homologous WT proteins. N-specific (C) and S-specific (D) activation of T cells in splenocytes from mice immunized with the candidate vaccine or the inactivated vaccine at 14 days after the third immunization. N-specific (E) and S-specific (F) activation of T cells in lung tissues from mice immunized with the candidate vaccine or the inactivated vaccine at 14 days after the third immunization (n = 4 mice per group). Significance was determined via one-way ANOVA with a Tukey multiple comparison test. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ns, no significance.

Article Snippet: B.1.351 variant N protein , SinoBiological , Cat# 40588-V07E9.

Techniques: Neutralization, Variant Assay, Incubation, Transduction, Luciferase, Activity Assay, Activation Assay

Journal: Cell Reports

Article Title: A two-adjuvant multiantigen candidate vaccine induces superior protective immune responses against SARS-CoV-2 challenge

doi: 10.1016/j.celrep.2021.110112

Figure Lengend Snippet:

Article Snippet: B.1.351 variant N protein , SinoBiological , Cat# 40588-V07E9.

Techniques: Blocking Assay, Recombinant, Variant Assay, Enzyme-linked Immunospot, Luciferase, Reporter Assay, Enzyme-linked Immunosorbent Assay, Transgenic Assay, Software, Filtration, Modification

Journal: Frontiers in Microbiology

Article Title: Production of SARS-CoV-2 N Protein-Specific Monoclonal Antibody and Its Application in an ELISA-Based Detection System and Targeting the Interaction Between the Spike C-Terminal Domain and N Protein

doi: 10.3389/fmicb.2021.726231

Figure Lengend Snippet: Production and characterization of the anti-SARS-CoV-2 N protein-specific monoclonal antibody. (A) Expression of biotin peptide-6 × His-tagged SARS-CoV-2 N protein (SARS-CoV-2 N-Bio-His 6 ). Recombinant SARS-CoV-2 N-Bio-His 6 protein was expressed in ExpiCHO cells and purified from cell culture supernatants using Ni-NTA agarose chromatography. The purified recombinant protein was analyzed by SDS-PAGE (left) and western blotting with peroxidase-conjugated streptavidin (middle) and anti-His-tag antibody (right). Arrowhead, biotin peptide-6 × His-tagged SARS-CoV-2 N protein; R, reducing condition; NR, non-reducing condition. (B) Expression of biotin peptide-6 × His-tagged MERS-CoV N protein (MERS-CoV N-Bio-His 6 ). The purified recombinant protein was analyzed by SDS-PAGE. Arrowhead, biotin peptide-6 × His-tagged MERS-CoV N protein. (C) The recombinant SARS-CoV-2 N-Bio-His 6 protein and CpG-DNA were combined in a DOPE:CHEMS complex and the complex was injected intraperitoneally into BALB/c mice ( n = 4) three times. ELISA was performed with mouse sera to determine whether recombinant SARS-CoV-2 N-Bio-His 6 protein-specific antibody was present. (D) Ascites were collected from mice injected with cloned hybridoma cells (1G10C4). ELISA was performed with the ascites to determine whether recombinant SARS-CoV-2 N-Bio-His 6 protein-specific antibody was present. (E) The monoclonal antibody was purified from the ascitic fluid using Protein-A column chromatography and analyzed using SDS-PAGE. HC, heavy chain; LC, light chain. (F) Subclasses of the monoclonal antibody were identified by ELISA. (G) The detection limit of the monoclonal antibody against SARS-CoV-2 N-Bio-His 6 protein was measured by ELISA. (H) Binding of the monoclonal antibody to recombinant SARS-CoV-2 N-Bio-His 6 protein was measured by ELISA.

Article Snippet: For the immunoprecipitation assay, SARS-CoV- 2-, MERS- CoV-, or HCoV-OC43-infected Vero cell lysates were incubated with the SARS-CoV-2 N protein-specific monoclonal antibody (clone 1G10C4 mAb) at 4 ° C for 2 h. Immunocomplexes were isolated with Protein A beads (Repligen, Waltham, MA, United States) and analyzed by western blotting with rabbit anti-SARS-CoV-2 N protein polyclonal antibody (Catalog No. 40588-T62; Sino Biological, Vienna, Austria).

Techniques: Expressing, Recombinant, Purification, Cell Culture, Chromatography, SDS Page, Western Blot, Injection, Enzyme-linked Immunosorbent Assay, Clone Assay, Column Chromatography, Binding Assay

Journal: Frontiers in Microbiology

Article Title: Production of SARS-CoV-2 N Protein-Specific Monoclonal Antibody and Its Application in an ELISA-Based Detection System and Targeting the Interaction Between the Spike C-Terminal Domain and N Protein

doi: 10.3389/fmicb.2021.726231

Figure Lengend Snippet: Specificity of the anti-SARS-CoV-2 N protein monoclonal antibody. (A) Analysis of monoclonal antibody specificity. Recombinant SARS-CoV-2 N-Bio-His 6 protein or recombinant MERS-CoV N-Bio-His 6 protein was captured on streptavidin-coated 96-well immunoplates and then incubated with anti-SARS-CoV-2 N protein monoclonal antibody. Reactivity of the monoclonal antibody to each recombinant protein was determined by ELISA. (B) MERS- CoV-, SARS-CoV- 2-, or HCoV-OC43-infected and non-infected Vero cell lysates were immunoblotted with the anti-SARS-CoV-2 N protein monoclonal antibody (clone 1G10C4 mAb). β-Actin was used as the loading control. (C) MERS- CoV-, SARS-CoV- 2-, or HCoV-OC43-infected and non-infected Vero cell lysates were immunoprecipitated with normal mouse IgG or the anti-SARS-CoV-2 N protein monoclonal antibody (clone 1G10C4 mAb). The immunocomplexes were subjected to western blot analysis using rabbit anti-SARS-CoV-2 N protein antibody (Catalog No. 40588-T62; Sino Biological).

Article Snippet: For the immunoprecipitation assay, SARS-CoV- 2-, MERS- CoV-, or HCoV-OC43-infected Vero cell lysates were incubated with the SARS-CoV-2 N protein-specific monoclonal antibody (clone 1G10C4 mAb) at 4 ° C for 2 h. Immunocomplexes were isolated with Protein A beads (Repligen, Waltham, MA, United States) and analyzed by western blotting with rabbit anti-SARS-CoV-2 N protein polyclonal antibody (Catalog No. 40588-T62; Sino Biological, Vienna, Austria).

Techniques: Recombinant, Incubation, Enzyme-linked Immunosorbent Assay, Infection, Immunoprecipitation, Western Blot

Journal: Frontiers in Microbiology

Article Title: Production of SARS-CoV-2 N Protein-Specific Monoclonal Antibody and Its Application in an ELISA-Based Detection System and Targeting the Interaction Between the Spike C-Terminal Domain and N Protein

doi: 10.3389/fmicb.2021.726231

Figure Lengend Snippet: Detection of N protein in cells infected with SARS-CoV-2 S, GH, or GR clade with anti-SARS-CoV-2 N protein-specific monoclonal antibody. (A,B) Western blot analyses for the detection of N protein in cells infected with SARS-CoV-2 S, GH, or GR clade. Vero cells (A) and Calu-3 cells (B) were infected with SARS-CoV-2 S, GH, or GR clade at a MOI of 0.1 for 72 h. Cell lysates were analyzed by western blotting with the anti-SARS-CoV-2 N protein-specific monoclonal antibody (clone 1G10C4 mAb). β-Actin was used as the loading control. (C) Confocal microscopy was used to detect N protein in cells infected with SARS-CoV-2 S, GH, or GR clade. Vero cells were cultured on cover glass in 12-well plates and infected with SARS-CoV-2 S, GH, or GR clade at a MOI of 0.1 for 48 h. The infected Vero cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. The cells were incubated with anti-SARS-CoV-2 N protein-specific monoclonal antibody or normal IgG and then with Alexa Fluor 488–conjugated goat anti-mouse IgG. Nuclei were stained with Hoechst 33258. Images were obtained by confocal microscope. Scale bar, 10 μm.

Article Snippet: For the immunoprecipitation assay, SARS-CoV- 2-, MERS- CoV-, or HCoV-OC43-infected Vero cell lysates were incubated with the SARS-CoV-2 N protein-specific monoclonal antibody (clone 1G10C4 mAb) at 4 ° C for 2 h. Immunocomplexes were isolated with Protein A beads (Repligen, Waltham, MA, United States) and analyzed by western blotting with rabbit anti-SARS-CoV-2 N protein polyclonal antibody (Catalog No. 40588-T62; Sino Biological, Vienna, Austria).

Techniques: Infection, Western Blot, Confocal Microscopy, Cell Culture, Incubation, Staining, Microscopy

Journal: Frontiers in Microbiology

Article Title: Production of SARS-CoV-2 N Protein-Specific Monoclonal Antibody and Its Application in an ELISA-Based Detection System and Targeting the Interaction Between the Spike C-Terminal Domain and N Protein

doi: 10.3389/fmicb.2021.726231

Figure Lengend Snippet: Detection of N protein in SARS-CoV-2 S, GH, or GR clade virus particles with anti-SARS-CoV-2 N protein-specific monoclonal antibody. (A) Western blot analyses. Vero cells were infected with SARS-CoV-2 S, GH, or GR clade at a MOI of 0.01 for 72 h and then cell culture supernatants were collected. The cell culture supernatants were lysed with cell lysis buffer and analyzed by western blotting with the anti-SARS-CoV-2 N protein-specific monoclonal antibody (clone 1G10C4 mAb). Virus titers were measured by plaque assay. (B) Immunoprecipitation analysis. Vero cells were infected with SARS-CoV-2 S, GH, or GR clade at a MOI of 0.01 for 72 h and then cell culture supernatants were collected. The cell culture supernatants were lysed with cell lysis buffer and immunoprecipitated with normal mouse IgG or the anti-SARS-CoV-2 N protein monoclonal antibody (clone 1G10C4 mAb). The immunocomplexes were subjected to western blot analysis using rabbit anti-SARS-CoV-2 N protein antibody (Catalog No. 40588-T62; Sino Biological).

Article Snippet: For the immunoprecipitation assay, SARS-CoV- 2-, MERS- CoV-, or HCoV-OC43-infected Vero cell lysates were incubated with the SARS-CoV-2 N protein-specific monoclonal antibody (clone 1G10C4 mAb) at 4 ° C for 2 h. Immunocomplexes were isolated with Protein A beads (Repligen, Waltham, MA, United States) and analyzed by western blotting with rabbit anti-SARS-CoV-2 N protein polyclonal antibody (Catalog No. 40588-T62; Sino Biological, Vienna, Austria).

Techniques: Western Blot, Infection, Cell Culture, Lysis, Plaque Assay, Immunoprecipitation

Journal: Frontiers in Microbiology

Article Title: Production of SARS-CoV-2 N Protein-Specific Monoclonal Antibody and Its Application in an ELISA-Based Detection System and Targeting the Interaction Between the Spike C-Terminal Domain and N Protein

doi: 10.3389/fmicb.2021.726231

Figure Lengend Snippet: N protein in SARS-CoV-2 S, GH, or GR clade virus particles interact with SARS-CoV-2 Spike CD. (A) Expression of SARS-CoV-2 Spike CD-Fc. The recombinant Fc control protein and the SARS-CoV-2 Spike CD-Fc fusion protein were expressed in ExpiCHO cells, purified from cell culture supernatants using Protein A column chromatography and analyzed by SDS-PAGE and Coomassie blue staining. (B) Interaction of the N protein in virus particles with SARS-CoV-2 Spike CD. Vero cells were infected with SARS-CoV-2 S, GH, or GR clade at a MOI of 0.01 for 72 h and then cell culture supernatants were collected. Cell culture supernatants were lysed with cell lysis buffer and incubated with Fc or SARS-CoV-2 Spike CD-Fc. Fc-bound proteins were pulled down with Protein A beads and subjected to western blot analysis using the anti-SARS-CoV-2 N protein-specific monoclonal antibody (clone 1G10C4 mAb). Western blot of virus lysates is shown as a control.

Article Snippet: For the immunoprecipitation assay, SARS-CoV- 2-, MERS- CoV-, or HCoV-OC43-infected Vero cell lysates were incubated with the SARS-CoV-2 N protein-specific monoclonal antibody (clone 1G10C4 mAb) at 4 ° C for 2 h. Immunocomplexes were isolated with Protein A beads (Repligen, Waltham, MA, United States) and analyzed by western blotting with rabbit anti-SARS-CoV-2 N protein polyclonal antibody (Catalog No. 40588-T62; Sino Biological, Vienna, Austria).

Techniques: Expressing, Recombinant, Purification, Cell Culture, Column Chromatography, SDS Page, Staining, Infection, Lysis, Incubation, Western Blot

Journal: Frontiers in Microbiology

Article Title: Production of SARS-CoV-2 N Protein-Specific Monoclonal Antibody and Its Application in an ELISA-Based Detection System and Targeting the Interaction Between the Spike C-Terminal Domain and N Protein

doi: 10.3389/fmicb.2021.726231

Figure Lengend Snippet: Interaction between SARS-CoV-2 Spike CD-Fc and SARS-CoV-2 N-Bio-His 6 recombinant proteins. (A) Schematic of the bait and prey assay system. (B) Immunoplates (96-well) were coated with streptavidin and then recombinant SARS-CoV-2 N-Bio-His 6 protein was added to each well. After addition of MERS-CoV Spike CD-Fc or SARS-CoV-2 Spike CD-Fc, the amount of CoV Spike CD-human Fc fusion protein bound to recombinant SARS-CoV-2 N-Bio-His 6 protein in the wells was determined by ELISA. (C) Specificity of the interaction between SARS-CoV-2 Spike CD-Fc and SARS-CoV-2 N-Bio-His 6 recombinant protein. Serially diluted non-biotinylated-recombinant SARS-CoV-2 N-His 6 protein was incubated with SARS-CoV-2 Spike CD-Fc and then added to the wells containing SARS-CoV-2 N-Bio-His 6 -coated streptavidin. The extent of the competition was measured by ELISA using HRP-conjugated anti-human IgG Fc antibody.

Article Snippet: For the immunoprecipitation assay, SARS-CoV- 2-, MERS- CoV-, or HCoV-OC43-infected Vero cell lysates were incubated with the SARS-CoV-2 N protein-specific monoclonal antibody (clone 1G10C4 mAb) at 4 ° C for 2 h. Immunocomplexes were isolated with Protein A beads (Repligen, Waltham, MA, United States) and analyzed by western blotting with rabbit anti-SARS-CoV-2 N protein polyclonal antibody (Catalog No. 40588-T62; Sino Biological, Vienna, Austria).

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Incubation

Journal: Frontiers in Microbiology

Article Title: Production of SARS-CoV-2 N Protein-Specific Monoclonal Antibody and Its Application in an ELISA-Based Detection System and Targeting the Interaction Between the Spike C-Terminal Domain and N Protein

doi: 10.3389/fmicb.2021.726231

Figure Lengend Snippet: Detection of SARS-CoV-2 in cell culture media using SARS-CoV-2 N protein-specific monoclonal antibody and recombinant SARS-CoV-2 Spike CD-Fc protein. (A) Schematic of the ELISA. (B) SARS-CoV-2 clade S and HCoV-OC43 and (C) SARS-CoV-2 clade GH and clade GR in cell culture supernatants were lysed with cell lysis buffer and serially diluted in PBST. These virus lysates were added into 96-well immunoplates coated with SARS-CoV-2 N protein-specific monoclonal antibody. After incubation for 2 h at room temperature, recombinant SARS-CoV-2 Spike CD-Fc protein was added, and then HRP-conjugated anti-human IgG Fc antibody was added to each well. The amount of SARS-CoV-2 N protein in each well was determined by ELISA.

Article Snippet: For the immunoprecipitation assay, SARS-CoV- 2-, MERS- CoV-, or HCoV-OC43-infected Vero cell lysates were incubated with the SARS-CoV-2 N protein-specific monoclonal antibody (clone 1G10C4 mAb) at 4 ° C for 2 h. Immunocomplexes were isolated with Protein A beads (Repligen, Waltham, MA, United States) and analyzed by western blotting with rabbit anti-SARS-CoV-2 N protein polyclonal antibody (Catalog No. 40588-T62; Sino Biological, Vienna, Austria).

Techniques: Cell Culture, Recombinant, Enzyme-linked Immunosorbent Assay, Lysis, Incubation

Journal: Frontiers in Immunology

Article Title: Circulating microRNA signatures associated with disease severity and outcome in COVID-19 patients

doi: 10.3389/fimmu.2022.968991

Figure Lengend Snippet: Modelling SARS-CoV-2 infection and IFNα stimulation in different human cell types. (A) Representative confocal microscopy images of lung carcinoma epithelial cells Calu-3, colon carcinoma epithelial cells Caco-2, human umbilical vein endothelial cells HUVEC, and peripheral blood mononuclear cells (PBMC) infected with SARS-CoV-2 or mock infected. Cells were stained with anti-SARS-CoV-2 nucleoprotein antibody (red) at 24 hours post infection. Nuclei were stained with draq5 (blue). 20× magnification. (B) Kinetics of SARS-CoV-2 replication in Calu-3, Caco-2, HUVEC, and PBMC. Viral load was measured by TCID50 assay in cell culture supernatant collected at different hours post infection (hpi) with SARS-CoV-2 at MOI 0.1 or 2. Viral titer is represented as mean ± SD of Log TCID50 values obtained from two experiments conducted in triplicate. (C) Expression of the IFN stimulated genes IFIT1 and IFIT2 , the pro-inflammatory cytokine genes IL6 and IL1B , and the ssRNA sensor genes TLR7 and TLR8 in HUVEC, PBMC, Caco-2 and Calu-3 cells at 24 hpi with SARS-CoV-2 at MOI 01 or 2 or treatment with IFN-α2b 1000 U/mL. mRNA expression was measured by real-time RT-PCR and represented as mean ± SD of log2 fold change vs. mock (calculated with the 2 -ΔΔCT method) obtained from two experiments conducted in triplicate. Comparison between groups (infected or treated cells vs. mock) was down by Mann-Whitney U test. *p<0.05.

Article Snippet: Immunofluorescence staining of SARS-CoV-2 Nucleocapsid protein was done with a rabbit monoclonal primary antibody (40143-R019; Sino Biological Inc., Beijing, China) at the dilution of 1:1000 and anti-rabbit IgG Alexa Fluor-546 secondary antibody (goat, 1:2000, Thermo Fisher Scientific).

Techniques: Infection, Confocal Microscopy, Staining, TCID50 Assay, Cell Culture, Expressing, Quantitative RT-PCR, MANN-WHITNEY

Journal: Frontiers in Immunology

Article Title: Circulating microRNA signatures associated with disease severity and outcome in COVID-19 patients

doi: 10.3389/fimmu.2022.968991

Figure Lengend Snippet: MicroRNA expression following SARS-CoV-2 infection and IFNα stimulation in different human cell types. The miRNAs investigated in vitro were selected among the differentially expressed (9 upregulated and 9 downregulated) serum miRNAs identified by the study in COVID-19 patients vs. healthy controls (HC). (A) Heatmap representing baseline miRNAs expression in lung carcinoma epithelial cells Calu-3, colon carcinoma epithelial cells Caco-2, human umbilical vein endothelial cells HUVEC, and peripheral blood mononuclear cells (PBMC). Data represent -ΔC T values of miRNA normalized to the endogenous control RNU6B in triplicate samples. The color scale bar represents -ΔC T values. (B) Heatmap representing miRNA fold change in cells infected with SARS-CoV-2 at MOI 0.1 or 2 vs. mock infected cells at 6 h and 24 h post infection. mRNA expression was measured by real-time RT-PCR and represented as mean log2 fold change vs. mock (calculated with the 2 -ΔΔCT method) obtained from two experiments conducted in triplicate. The color scale bar represents mean log2 fold change vs. mock. (C) Heatmap representing miRNA fold change in cells treated for 24 h with IFNα 1000 U/mL vs. mock treated cells. mRNA expression was measured by real-time RT-PCR and represented as mean log2 fold change vs. mock (calculated with the 2 -ΔΔCT method) obtained from two experiments conducted in triplicate. The color scale bar represents mean log2 fold change vs. mock.

Article Snippet: Immunofluorescence staining of SARS-CoV-2 Nucleocapsid protein was done with a rabbit monoclonal primary antibody (40143-R019; Sino Biological Inc., Beijing, China) at the dilution of 1:1000 and anti-rabbit IgG Alexa Fluor-546 secondary antibody (goat, 1:2000, Thermo Fisher Scientific).

Techniques: Expressing, Infection, In Vitro, Quantitative RT-PCR

Journal: Frontiers in Immunology

Article Title: Circulating microRNA signatures associated with disease severity and outcome in COVID-19 patients

doi: 10.3389/fimmu.2022.968991

Figure Lengend Snippet: MicroRNAs modulated in COVID-19 patients. (A) Illustration of the study design and results, highlighting relevant serum miRNAs that were differentially expressed between COVID-19 patients vs. HC and between severe COVID-19 vs. mild and moderate COVID-19. The figure also shows serum miRNAs significantly associated with the risk of intensive care unit (ICU) hospitalization and death in COVID-19 patients, as well as male sex and age. (B) Illustration of the results of in vitro experiments, summarizing the effects of SARS-CoV-2 infection and IFN-α2b treatment on human lung (Calu-3), colon (Caco-2), endothelial (HUVEC), and peripheral blood mononuclear cells (PBMCs). Upregulated and downregulated miRNAs are represented in red and blue, respectively.

Article Snippet: Immunofluorescence staining of SARS-CoV-2 Nucleocapsid protein was done with a rabbit monoclonal primary antibody (40143-R019; Sino Biological Inc., Beijing, China) at the dilution of 1:1000 and anti-rabbit IgG Alexa Fluor-546 secondary antibody (goat, 1:2000, Thermo Fisher Scientific).

Techniques: In Vitro, Infection