Journal: Oncotarget

Article Title: Epidermal growth factor receptor mutation mediates cross-resistance to panitumumab and cetuximab in gastrointestinal cancer

doi:

Figure Lengend Snippet: NGS of EGFR axons 7-13, KRAS exons 2/3/4 and NRAS exons 2/3/4 in pre- and post-treatment samples from the "tumor tissue" patient cohort. *

Article Snippet: Transfected CHO or Ba/F3 cells were stained with a polyclonal goat anti-human EGFR antibody (R&D Systems, Minneapolis, USA), panitumumab or cetuximab followed by secondary detection with FITC-labeled rabbit anti-human (Sigma-Aldrich, St.Gallen, Switzerland), or rabbit anti-goat antibodies (Dako Cytomation, Copenhagen, Denmark).

Techniques: Control

Journal: Oncotarget

Article Title: Epidermal growth factor receptor mutation mediates cross-resistance to panitumumab and cetuximab in gastrointestinal cancer

doi:

Figure Lengend Snippet: A: Localization of EGFR mutations G465R and S492R on the ectodomain of the EGFR . The 3-dimensional EGFR model was created from pdb file 1NQL from RCSB Protein Data Bank. The S492 position is shown in blue, the G465 position in black. The panel on the right shows the EGFR domain III alone with panitumumab epitope in red and cetuximab epitope in blue. Overlaps of mutated positions G465 and S492 with antibody epitopes are shown. B: EGFR mutation G465R abrogates binding of panitumumab and cetuximab at the protein level. Wile-type and mutant EGFR-Fc proteins were expressed and the binding of therapeutic antibodies to immobilized proteins was assessed by ELISA. Data are means from 3 experiments +/− SEM. * p<0.05, ** p<0.01, n.s. = not significant (student's T-test comparing binding of respective antibody to mutant versus wt EGFR-Fc) C and D: Panitumumab and cetuximab binding is abrogated in CHO cells transfected with EGFR G465R . EGFR negative CHO cells were transfected with wild type EGFR or mutants thereof. Binding of panitumumab, cetuximab or a control polyclonal EGFR antibody was assessed by FACS analysis 48h after transfection. FSC = forward scatter. Panel C shows exemplary FACS plots, panel D shows mean data from 5 experiments with binding of the polyclonal EGFR antibody set to 100% +/− SEM. * p<0.05, ** p<0.01, n.s. = not significant (student's T-test comparing binding of respective antibody to mutant versus wt EGFR).

Article Snippet: Transfected CHO or Ba/F3 cells were stained with a polyclonal goat anti-human EGFR antibody (R&D Systems, Minneapolis, USA), panitumumab or cetuximab followed by secondary detection with FITC-labeled rabbit anti-human (Sigma-Aldrich, St.Gallen, Switzerland), or rabbit anti-goat antibodies (Dako Cytomation, Copenhagen, Denmark).

Techniques: Mutagenesis, Binding Assay, Enzyme-linked Immunosorbent Assay, Transfection, Control

Journal: Oncotarget

Article Title: Epidermal growth factor receptor mutation mediates cross-resistance to panitumumab and cetuximab in gastrointestinal cancer

doi:

Figure Lengend Snippet: A: EGFR signaling in EGF-dependent Ba/F3 model. Wt and S492R or G465R mutant EGFR-expressing Ba/F3 cells were cultured in the presence or absence of EGF and with addition of cetuximab, panitumumab, rituximab or erlotinib. After 2 hours, cells were harvested and EGFR/pEGFR expression analyzed by western blot analysis. B: Sensitivity of EGFR wt or EGFR G465R mutant-transfected Ba/F3 cells to treatment with EGFR-targeted antibodies. Ba/F3 cells were transformed to IL-3 independence with EGFR wt or mutant constructs and subsequently cultured in the presence or absence of EGF or with EGF in combination with panitumumab, cetuximab or control antibody rituximab. The number of viable cells was determined by trypan blue exclusion every 12 hours beginning 24 hours after seeding and plotted. Data are means from triplicate experiments +/−SEM.

Article Snippet: Transfected CHO or Ba/F3 cells were stained with a polyclonal goat anti-human EGFR antibody (R&D Systems, Minneapolis, USA), panitumumab or cetuximab followed by secondary detection with FITC-labeled rabbit anti-human (Sigma-Aldrich, St.Gallen, Switzerland), or rabbit anti-goat antibodies (Dako Cytomation, Copenhagen, Denmark).

Techniques: Mutagenesis, Expressing, Cell Culture, Western Blot, Transfection, Transformation Assay, Construct, Control

Journal: Oncotarget

Article Title: Epidermal growth factor receptor mutation mediates cross-resistance to panitumumab and cetuximab in gastrointestinal cancer

doi:

Figure Lengend Snippet: Clinical characteristics of the "liquid biopsy" patient cohort subjected to NGS of circulating tumor DNA. *

Article Snippet: Transfected CHO or Ba/F3 cells were stained with a polyclonal goat anti-human EGFR antibody (R&D Systems, Minneapolis, USA), panitumumab or cetuximab followed by secondary detection with FITC-labeled rabbit anti-human (Sigma-Aldrich, St.Gallen, Switzerland), or rabbit anti-goat antibodies (Dako Cytomation, Copenhagen, Denmark).

Techniques: Control, Clinical Proteomics

Journal: Oncotarget

Article Title: Epidermal growth factor receptor mutation mediates cross-resistance to panitumumab and cetuximab in gastrointestinal cancer

doi:

Figure Lengend Snippet: EGFR G465R and S492R ectodomain mutations and also KRAS and NRAS mutations after EGFR antibody treatment in circulating tumor DNA from the "liquid biopsy” patient cohort. *

Article Snippet: Transfected CHO or Ba/F3 cells were stained with a polyclonal goat anti-human EGFR antibody (R&D Systems, Minneapolis, USA), panitumumab or cetuximab followed by secondary detection with FITC-labeled rabbit anti-human (Sigma-Aldrich, St.Gallen, Switzerland), or rabbit anti-goat antibodies (Dako Cytomation, Copenhagen, Denmark).

Techniques: Mutagenesis, Control

Journal: Cell

Article Title: Generation of a Broadly Useful Model for COVID-19 Pathogenesis, Vaccination, and Treatment

doi: 10.1016/j.cell.2020.06.010

Figure Lengend Snippet: Development of Mice Sensitized to SARS-CoV-2 Infection (A and B) To assess hACE2 expression and surface localization, 17CL-1 cells were transduced with Ad5-hACE2 or Ad5-Empty at MOI of 100 at 37°C for 4 h. hACE2 expression was monitored by western blot assay (A) or flow cytometry (B). (C) Ad5-hACE2 transduced 17CL-1 cells were infected with SARS-CoV-2 at MOI of 0.5 at 48 h post transduction, and virus titers were determined by foci forming assay (FFA) at 24, 48, and 72 hours post infection (h.p.i.). (D) Five days after transduction with 2.5 × 10 8 FFU of Ad5-hACE2 or Ad5-Empty in 75 μL of DMEM intranasally, lungs were harvested from BALB/c mice, fixed in zinc formalin, and embedded in paraffin. Sections were stained with an anti-hACE2 antibody (brown color). hACE2 protein (brown color) was detected only in Ad-hACE2-treated mice and was predominantly localized to alveolar epithelial cells. Scale bars, 467 and 94 μm, top and bottom panels, respectively. (E and F) Ad5-hACE2- or Ad5-Empty-transduced BALB/c or C57BL/6 mice were intranasally infected with 1 × 10 5 PFU of SARS-CoV-2 in 50 μL of DMEM. Weight changes in 6-to-8-week old BALB/c (E) and C57BL/6 (F) mice were monitored daily (n = 5 mice per group). To obtain virus kinetics in BALB/c (E) and C57BL/6 (F) mice, lungs were harvested and homogenized at the indicated time points, and virus was titered by plaque assay. Titers are expressed as PFU/g lung tissue (n = 3 mice per group per time point). Data are representative of two independent experiments. (G) 2 d.p.i., lungs were harvested from BALB/c mice, fixed in zinc formalin, and embedded in paraffin. Sections were stained with anti-SARS-CoV-2 N protein. Scale bar, 476 μm. (H) Representative Hematoxylin-eosin (HE) staining of lungs from BALB/c and C57BL/6 mice harvested at the indicated time points p.i. Scale bars, 443 and 88 μm, top and bottom panels, respectively. Asterisk, edema. (I) Summary histology scores determined at the indicated time points (n = 4 to 5 mice per group). PMN, neutrophils. (J) Photographs of lung specimens isolated from infected mice at indicated time points are shown. Arrowheads indicate regions with vascular congestion and hemorrhage.

Article Snippet: Cells were then incubated with a rabbit anti-SARS-CoV-2 nucleocapsid protein polyclonal antibody (Cat. No.: 40143-T62, Sino Biological, Inc. Beijing), followed by an HRP-labeled goat anti-rabbit secondary antibody (Cat. No.: 109-035-088, Jackson ImmunoResearch Laboratories, Inc. West Grove, PA).

Techniques: Infection, Expressing, Transduction, Western Blot, Flow Cytometry, Staining, Plaque Assay, Isolation

Journal: Cell

Article Title: Generation of a Broadly Useful Model for COVID-19 Pathogenesis, Vaccination, and Treatment

doi: 10.1016/j.cell.2020.06.010

Figure Lengend Snippet: The Role for IFN and STAT1 Signaling in SARS-CoV-2 Infection (A) 5 days after transduction with 2.5 × 10 8 FFU of Ad5-hACE2, C57BL/6 mice were intranasally infected with 1 × 10 5 PFU of SARS-CoV-2. Weight changes were monitored daily (n = 5 mice per group), and virus titers in the lungs were measured at the indicated time points using FFA (n = 3–4 mice per group per time point). Titers are expressed as FFU/g tissue. (B) Sections of paraffin embedded lungs from SARS-CoV-2-infected Ad5-hACE2-transduced, wild-type and genetically modified C57BL/6 mice at 4 d.p.i. were stained with hematoxylin/eosin. Scale bar, 100 μm. (C) Photographs of gross pathological lung specimens isolated from infected C57BL/6 mice at 4 d.p.i. Arrowheads indicate regions with vascular congestion and hemorrhage. (D) Ad5-hACE2-transduced C57BL/6 mice were treated with 80 μg of poly I:C in 50 μL of PBS 6 h before intranasal infection with SARS-CoV-2. Weight changes were monitored daily, and viral titers in lungs were measured at the indicated time points. ∗ p values ≤ 0.05; ∗∗ p values ≤ 0.005; ∗∗∗ p values ≤ 0.0005; ∗∗∗∗ p values ≤ 0.0001.

Article Snippet: Cells were then incubated with a rabbit anti-SARS-CoV-2 nucleocapsid protein polyclonal antibody (Cat. No.: 40143-T62, Sino Biological, Inc. Beijing), followed by an HRP-labeled goat anti-rabbit secondary antibody (Cat. No.: 109-035-088, Jackson ImmunoResearch Laboratories, Inc. West Grove, PA).

Techniques: Infection, Transduction, Genetically Modified, Staining, Isolation

Journal: Cell

Article Title: Generation of a Broadly Useful Model for COVID-19 Pathogenesis, Vaccination, and Treatment

doi: 10.1016/j.cell.2020.06.010

Figure Lengend Snippet: Differentially Expressed Genes in the Lungs of SARS-CoV-2-Infected Mice (A) SARS-CoV-2 viral RNA detected by RNA-seq in Ad5-Empty- and Ad5-ACE2-transduced mouse lungs. Data are expressed as normalized read counts. (B) Volcano plot showing differentially expressed genes in the lungs of Ad5-ACE2-transduced mice compared with Ad5-Empty-transduced mice. A total of 3,056 transcripts were differentially regulated. (C) Gene ontology (GO) analysis showing the differentially expressed genes from (B). (D) CD4 + (Cd4), CD8 + (Cd8a) T cell and B cell (Cd79b), macrophage (Cd68), and monocyte (Cd14) lineage marker expression. The red lines are the means of the three biological replicates, and the error bars are the standard error of the mean. Data are expressed as normalized read counts. p values are from a one-tailed Student’s t test. (E) Selected cytokines and chemokines differentially regulated in the lungs of Ad5-Empty- and Ad5-ACE2-transduced BALB/c mice at 2 d.p.i., obtained from the RNA-seq data. The red lines are the means of the three biological replicates, and the error bars are the standard error of the mean. Data are expressed as normalized read counts. p values are from a one-tailed Student’s t test.

Article Snippet: Cells were then incubated with a rabbit anti-SARS-CoV-2 nucleocapsid protein polyclonal antibody (Cat. No.: 40143-T62, Sino Biological, Inc. Beijing), followed by an HRP-labeled goat anti-rabbit secondary antibody (Cat. No.: 109-035-088, Jackson ImmunoResearch Laboratories, Inc. West Grove, PA).

Techniques: Infection, RNA Sequencing Assay, Marker, Expressing, One-tailed Test

Journal: Cell

Article Title: Generation of a Broadly Useful Model for COVID-19 Pathogenesis, Vaccination, and Treatment

doi: 10.1016/j.cell.2020.06.010

Figure Lengend Snippet: Requirements for T Cells and Antibodies for SARS-CoV-2 Clearance and Protection from Subsequent Challenge Ad5-hACE2-transduced mice were infected with 1 × 10 5 PFU of SARS-CoV-2. (A) For systemic depletion of CD4 + or CD8 + T cells, mice were injected intraperitoneally (i.p.) with 0.5 mg anti-CD4 antibody (clone GK1.5) and/or 0.5 mg anti-CD8 antibody (clone 2.43) or 0.5 mg rat IgG at days −2 and 0 p.i. Virus titers in the lungs were measured at the indicated time points. Titers are expressed as FFU/g tissue (n = 4 mice per group per time point) (B–D) To identify SARS-CoV-2 T cell responses, single-cell suspensions were prepared from the BALF of transduced/infected BALB/c mice and stimulated with 2 μM structural protein peptide pools for 5–6 h in the presence of brefeldin A. Flow plots (B, 7 d.p.i), and summary of frequencies and cell numbers of SARS-CoV-2-N pool specific CD4 + T cells (C) and S1 pool specific CD8 + T cells (D) (determined by IFN-γ intracellular staining) are shown (n = 3 to 4 mice per time point). (E) PRNT 50 titers in the sera of transduced/infected C57BL/6 mice at indicated time points p.i. are shown. (F) BALB/c and C57BL/6 mice were immunized with 1 × 10 5 infectious units (IU) of VRP-S intranasally in 50 μL of PBS. Mice were transduced and infected with 1 × 10 5 PFU of SARS-CoV-2 3 weeks after vaccination. Virus titers in the lungs were measured at the indicated time points (n = 4 mice per group per time point). (G) For adoptive transfer of serum, BALB/c mice were immunized with 1 × 10 5 IU of VRP in the footpad in 50 μL of PBS and boosted with the same dose 3 weeks later. Sera were obtained 1–2 weeks after VRP booster. Then, 150 μL of serum was transferred into transduced mice intravenously (i.v.) 1 day before SARS-CoV-2 infection (n = 3 mice per group per time point). ∗ p values ≤ 0.05; ∗∗ p values ≤ 0.005; ∗∗∗ p values ≤ 0.0005; ∗∗∗∗ p values ≤ 0.0001.

Article Snippet: Cells were then incubated with a rabbit anti-SARS-CoV-2 nucleocapsid protein polyclonal antibody (Cat. No.: 40143-T62, Sino Biological, Inc. Beijing), followed by an HRP-labeled goat anti-rabbit secondary antibody (Cat. No.: 109-035-088, Jackson ImmunoResearch Laboratories, Inc. West Grove, PA).

Techniques: Infection, Injection, Staining, Adoptive Transfer Assay

Journal: Cell

Article Title: Generation of a Broadly Useful Model for COVID-19 Pathogenesis, Vaccination, and Treatment

doi: 10.1016/j.cell.2020.06.010

Figure Lengend Snippet: Convalescent Plasma from COVID-19 Patients and Remdesivir Protect Mice from SARS-CoV-2 Infection (A and B) For plasma adoptive transfer, Ad5-hACE2-transduced mice were injected with 150 μL of plasma i.v. from a healthy donor or COVID-19, MERS, or SARS convalescent patients, at −1 d.p.i. Weight and virus titers in lung tissues were monitored (A) and expressed as FFU/g tissue (n = 4 mice per group per time point). Sections of paraffin embedded lungs from plasma adoptive transferred and infected mice were stained with HE at day 4 p.i. (B). Scale bar, 100 μm. (C and D) For remdesivir treatment, Ad5-hACE2-transduced mice were treated with remdesivir (25 mg/kg, bid s.c.) or vehicle at −1 d.p.i. Weight loss of infected mice and virus titers in the lungs were monitored (C), and hematoxylin/eosin staining of sections of paraffin-embedded lungs is shown at 4 d.p.i. (D) (n = 4 mice per group per time point). Data are representative of two independent experiments. Scale bar, 100 μm. ∗ p values ≤ 0.05; ∗∗ p values ≤ 0.005; ∗∗∗ p values ≤ 0.0005; ∗∗∗∗ p values of ≤ 0.0001.

Article Snippet: Cells were then incubated with a rabbit anti-SARS-CoV-2 nucleocapsid protein polyclonal antibody (Cat. No.: 40143-T62, Sino Biological, Inc. Beijing), followed by an HRP-labeled goat anti-rabbit secondary antibody (Cat. No.: 109-035-088, Jackson ImmunoResearch Laboratories, Inc. West Grove, PA).

Techniques: Infection, Adoptive Transfer Assay, Injection, Staining

Journal: Cell

Article Title: Generation of a Broadly Useful Model for COVID-19 Pathogenesis, Vaccination, and Treatment

doi: 10.1016/j.cell.2020.06.010

Figure Lengend Snippet:

Article Snippet: Cells were then incubated with a rabbit anti-SARS-CoV-2 nucleocapsid protein polyclonal antibody (Cat. No.: 40143-T62, Sino Biological, Inc. Beijing), followed by an HRP-labeled goat anti-rabbit secondary antibody (Cat. No.: 109-035-088, Jackson ImmunoResearch Laboratories, Inc. West Grove, PA).

Techniques: Blocking Assay, Plasmid Preparation, Software

Journal: NPJ Vaccines

Article Title: Modulating the immune response to SARS-CoV-2 by different nanocarriers delivering an mRNA expressing trimeric RBD of the spike protein: COVARNA Consortium

doi: 10.1038/s41541-024-00838-8

Figure Lengend Snippet: a Description of the different nanocarriers used for the encapsulation of RBD-mRNA. b Detection of RBD expression in cellular pellets and supernatants from 293T cells transfected with the different formulated RBD-mRNAs for 6 h by western-blotting analysis using a rabbit polyclonal anti-SARS-CoV-2 spike/RBD antibody (upper panels). Ponceau staining (lower panels) was used as loading control. All blots derive from the same experiment and were processed in parallel. c RBD expression and viability of human monocyte-derived dendritic cells (hMDDCs) from a healthy donor at 6 and 24 h after transfection with the different nanocarriers containing the RBD-mRNA. Mean with standard error of the mean (SEM) is represented.

Article Snippet: After centrifugation (1500 rpm, 5 min) and supernatant removal, cells were incubated with the live/dead fixable red dye (1:200; Invitrogen) at 4 °C in the dark for 30 min, washed twice with FACS buffer and fixed/permeabilized with BD Cytofix/Cytoperm (BD Biosciences, San Jose, CA, USA) at 4 °C for 20 min. After, cells were centrifuged (1500 rpm, 5 min), washed twice with PermWash (PW) 1× buffer (diluted in FACS buffer; BD Biosciences) and blocked with PBS 1×-3% BSA at 4 °C for 30 min. Then, cells were incubated with a rabbit polyclonal anti-SARS-CoV-2 spike/RBD antibody (5 µg/mL; Sino Biological, Beijing, China) at 4 °C for 30 min in the dark.

Techniques: Encapsulation, Expressing, Transfection, Western Blot, Staining, Derivative Assay

Journal: NPJ Vaccines

Article Title: Modulating the immune response to SARS-CoV-2 by different nanocarriers delivering an mRNA expressing trimeric RBD of the spike protein: COVARNA Consortium

doi: 10.1038/s41541-024-00838-8

Figure Lengend Snippet: a Immunization schedule. Female C57BL/6 mice ( n = 5) were immunized with two doses of 40 µg of the different formulations containing RBD-mRNA by intramuscular (i.m.) route as indicated. b SARS-CoV-2 RBD-specific IgG binding antibodies. Anti-RBD IgG titers were determined in individual sera obtained at 20 days post-prime (d20) or 21 days post-boost (d42) by ELISA. An unpaired nonparametric Mann–Whitney test of transformed data was used. *** p < 0.001. c SARS-CoV-2 neutralizing antibody responses. NT 50 titers were determined in individual sera harvested at d20 and d42 using a live virus microneutralization assay (MAD6 strain, containing D614G mutation). An ordinary one-way ANOVA of transformed data followed by Tukey’s multiple comparison test was performed. *** p < 0.001. d Neutralizing antibody responses induced against SARS-CoV-2 variants. NT 50 titers were evaluated in individual serum samples harvested at d42 by a live virus microneutralization assay using the SARS-CoV-2 Alpha (B.1.1.7), Beta (B.1.351), Delta (B.1.617.2) and Omicron (B.1.1.529) variants. An ordinary one-way ANOVA of transformed data followed by Tukey’s multiple comparison test was performed. ** p < 0.01; *** p < 0.001. Serum samples from mice similarly vaccinated with two doses of 5 μg of BNT162b2 vaccine (mRNA vaccine from Pfizer-BioNTech) were used as a reference value (BNT162b2 reference). Red dashed line represents the lower limit of detection of the assay. Mean with standard error of the mean (SEM) is represented.

Article Snippet: After centrifugation (1500 rpm, 5 min) and supernatant removal, cells were incubated with the live/dead fixable red dye (1:200; Invitrogen) at 4 °C in the dark for 30 min, washed twice with FACS buffer and fixed/permeabilized with BD Cytofix/Cytoperm (BD Biosciences, San Jose, CA, USA) at 4 °C for 20 min. After, cells were centrifuged (1500 rpm, 5 min), washed twice with PermWash (PW) 1× buffer (diluted in FACS buffer; BD Biosciences) and blocked with PBS 1×-3% BSA at 4 °C for 30 min. Then, cells were incubated with a rabbit polyclonal anti-SARS-CoV-2 spike/RBD antibody (5 µg/mL; Sino Biological, Beijing, China) at 4 °C for 30 min in the dark.

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY, Transformation Assay, Virus, Microneutralization Assay, Mutagenesis, Comparison

Journal: NPJ Vaccines

Article Title: Modulating the immune response to SARS-CoV-2 by different nanocarriers delivering an mRNA expressing trimeric RBD of the spike protein: COVARNA Consortium

doi: 10.1038/s41541-024-00838-8

Figure Lengend Snippet: a Immunization schedule. K18-hACE2 transgenic mice ( n = 6) were immunized with two doses of 40 µg of mLNP-RBD or LNP-1-RBD formulations by i.m. route as indicated. At day 47 mice were challenged intranasally (i.n.) with 1 × 10 5 PFU of SARS-CoV-2 (MAD6 isolate, containing D614G mutation). b SARS-CoV-2 S- and RBD-specific IgG binding antibodies. Anti-S and anti-RBD IgG titers were determined in individual sera obtained at 20 days post-prime (d20) or 21 days post-boost (d42) by ELISA. An unpaired nonparametric Mann-Whitney test of transformed data was used. * p < 0.05; ** p < 0.005; *** p < 0.001. c SARS-CoV-2 neutralizing antibody responses. NT 50 titers were determined in individual sera harvested at d20 and d42 using a live virus microneutralization assay (MAD6 strain, containing D614G mutation). An ordinary one-way ANOVA of transformed data followed by Tukey’s multiple comparison test was performed. ** p < 0.005. d Neutralizing antibody responses induced against SARS-CoV-2 variants. NT 50 titers were evaluated in individual sera collected at d42 by a live virus microneutralization assay using the SARS-CoV-2 Delta (B.1.617.2), Omicron (B.1.1.529), BQ1.1 and XBB1.5 variants. An ordinary one-way ANOVA of transformed data followed by Tukey’s multiple comparison test was performed. * p < 0.05; ** p < 0.005; *** p < 0.001. Serum samples from mice similarly vaccinated with two doses of 5 μg of BNT162b2 vaccine (mRNA vaccine from Pfizer-BioNTech) were used as a reference value (BNT162b2 reference). Red dashed line represents the lower limit of detection of the assay. Mean with standard error of the mean (SEM) is represented.

Article Snippet: After centrifugation (1500 rpm, 5 min) and supernatant removal, cells were incubated with the live/dead fixable red dye (1:200; Invitrogen) at 4 °C in the dark for 30 min, washed twice with FACS buffer and fixed/permeabilized with BD Cytofix/Cytoperm (BD Biosciences, San Jose, CA, USA) at 4 °C for 20 min. After, cells were centrifuged (1500 rpm, 5 min), washed twice with PermWash (PW) 1× buffer (diluted in FACS buffer; BD Biosciences) and blocked with PBS 1×-3% BSA at 4 °C for 30 min. Then, cells were incubated with a rabbit polyclonal anti-SARS-CoV-2 spike/RBD antibody (5 µg/mL; Sino Biological, Beijing, China) at 4 °C for 30 min in the dark.

Techniques: Transgenic Assay, Mutagenesis, Binding Assay, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY, Transformation Assay, Virus, Microneutralization Assay, Comparison

Journal: NPJ Vaccines

Article Title: Modulating the immune response to SARS-CoV-2 by different nanocarriers delivering an mRNA expressing trimeric RBD of the spike protein: COVARNA Consortium

doi: 10.1038/s41541-024-00838-8

Figure Lengend Snippet: Individual mice were daily monitored for changes of body weight ( a ) and mortality ( b ) for 14 days. Mice that lost more than 25% of the initial body weight were sacrificed. c Genomic ( RdRp ) and subgenomic ( N ) SARS-CoV-2 RNAs detected by RT-qPCR in lungs from individual mice at 14 days (groups 1 and 2) or 7 days (group 3) after SARS-CoV-2 challenge. Mean RNA copy numbers (copies/μl) with standard error of the mean (SEM) from duplicates of each lung sample is represented. Relative values are referred to uninfected mice (group 4). An ordinary one-way ANOVA of transformed data followed by Tukey’s multiple comparison test was performed. *** p < 0.001. d SARS-CoV-2 infectious virus in lung or nasal turbinates. Mean PFU (PFU/gram of lung tissue or PFU/mL of nasal turbinates) with SEM from triplicates of each sample is represented. An ordinary one-way ANOVA of transformed data followed by Tukey’s multiple comparison test was performed. ** p < 0.005; *** p < 0.001.

Article Snippet: After centrifugation (1500 rpm, 5 min) and supernatant removal, cells were incubated with the live/dead fixable red dye (1:200; Invitrogen) at 4 °C in the dark for 30 min, washed twice with FACS buffer and fixed/permeabilized with BD Cytofix/Cytoperm (BD Biosciences, San Jose, CA, USA) at 4 °C for 20 min. After, cells were centrifuged (1500 rpm, 5 min), washed twice with PermWash (PW) 1× buffer (diluted in FACS buffer; BD Biosciences) and blocked with PBS 1×-3% BSA at 4 °C for 30 min. Then, cells were incubated with a rabbit polyclonal anti-SARS-CoV-2 spike/RBD antibody (5 µg/mL; Sino Biological, Beijing, China) at 4 °C for 30 min in the dark.

Techniques: Quantitative RT-PCR, Transformation Assay, Comparison, Virus

Journal: NPJ Vaccines

Article Title: Modulating the immune response to SARS-CoV-2 by different nanocarriers delivering an mRNA expressing trimeric RBD of the spike protein: COVARNA Consortium

doi: 10.1038/s41541-024-00838-8

Figure Lengend Snippet: Proinflammatory cytokines and chemokines were detected by RT-qPCR in lungs from individual mice at 14 days (groups 1 and 2) or 7 days (group 3) after SARS-CoV-2 challenge. Mean RNA levels (in A.U.) with SEM from duplicates of each lung sample is represented; relative values are referred to uninfected mice (group 4). An ordinary one-way ANOVA of transformed data followed by Tukey’s multiple comparison test was performed. * p < 0.05; ** p < 0.005; *** p < 0.001.

Article Snippet: After centrifugation (1500 rpm, 5 min) and supernatant removal, cells were incubated with the live/dead fixable red dye (1:200; Invitrogen) at 4 °C in the dark for 30 min, washed twice with FACS buffer and fixed/permeabilized with BD Cytofix/Cytoperm (BD Biosciences, San Jose, CA, USA) at 4 °C for 20 min. After, cells were centrifuged (1500 rpm, 5 min), washed twice with PermWash (PW) 1× buffer (diluted in FACS buffer; BD Biosciences) and blocked with PBS 1×-3% BSA at 4 °C for 30 min. Then, cells were incubated with a rabbit polyclonal anti-SARS-CoV-2 spike/RBD antibody (5 µg/mL; Sino Biological, Beijing, China) at 4 °C for 30 min in the dark.

Techniques: Quantitative RT-PCR, Transformation Assay, Comparison