Journal: BMC Methods

Article Title: A dual-fluorescence assay for gene delivery vehicle screening in macrophages with an inflammation-inducible reporter construct

doi: 10.1186/s44330-025-00030-x

Figure Lengend Snippet: Characterization of RAW264.7-Inflammatory Reporter Cell line. A Overview of RAW264.7-IRC cell line creation. The inflammatory reporter construct, created using Gibson cloning, contained an mRFP1 fluorescent reporter under an inflammatory promoter, termed synthesis friendly inflammatory promoter (SFNp) , and was encapsulated and transduced via lentivirus. The cells then underwent puromycin selection to create a homogenous population of cells. RAW264.7-IRCs were then stimulated using different cytokines and inflammatory agents to induce mRFP1 expression. B LPS dose curve showing range of mRFP1 fluorescence geometric mean values measured using flow cytometry across a range of LPS doses from 0.001 to 100 ng/mL. Individual data points are plotted for each biological replicate. Each dose was done in triplicate. Trend line calculated using the three-parameter dose–response curve, which yielded an EC50 = 0.61 ng/mL with an R 2 value of 0.94. Dose–response curve line equation can be found in Table S1. C TNF-α concentration levels (pg/mL) in cell culture supernatant measured using ELISA. Cells were treated with LPS at concentrations ranging from 0.001 to 1 ng/mL for 24 h. Each data point represents an average of 2 technical replicates. Trend line calculated using the three-parameter dose–response curve, which yielded an EC50 = 0.87 ng/mL with an R 2 value of 0.987. Dose–response curve line equation can be found in Table S2 . D Plot of mRFP1 fluorescent geometric mean values plotted against respective TNF-α concentration levels (pg/mL). X -values represent the average of the 3 biological replicates of TNF-α concentration (from Fig. 1 C) and y -values represent the average of the 3 biological replicates of mRFP1 geometric mean (from Fig. 1B) ± SD. Trend line calculated using linear regression, y = 21.12 x −8156, with an R 2 value of 0.90. Linear regression parameters can be found in Table S3. E RAW264.7-IRC responsiveness. RAW264.7-IRCs were treated with LPS and IFN-γ for 24 h, 24 h cells were harvested for flow cytometry. Stimulation media was replaced with fresh media for the 48 h and 72 h samples. 24 h post-removal of stimulation media, the 48 h samples were harvested for flow cytometry, and the same was done for the 72 h samples. Mean ± SD, with individual data points for each biological replicate. (**** p -value ≤ 0.0001, *** p -value ≤ 0.001, ** p -value ≤ 0.01, n.s. p -value > 0.05). F Timeline of treatment for RAW264.7-IRC responsiveness

Article Snippet: The mRFP1 red fluorescent protein was taken from the pU6-pegRNA-GG-acceptor plasmid (Addgene #132,777) via PCR using Q5 High-Fidelity DNA Polymerase (NEB) and purified.

Techniques: Construct, Cloning, Selection, Expressing, Fluorescence, Flow Cytometry, Concentration Assay, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: BMC Methods

Article Title: A dual-fluorescence assay for gene delivery vehicle screening in macrophages with an inflammation-inducible reporter construct

doi: 10.1186/s44330-025-00030-x

Figure Lengend Snippet: Immunostaining for Inflammatory versus Non-Inflammatory Phenotype in RAW264.7-IRCs. A Phenotyping via immunostaining of RAW264.7-IRCs for CD206 (green), a common anti-inflammatory marker, and CD86 (blue), a common inflammatory marker, co-localized with mRFP1 expression between no treatment and LPS treated cells (10 ng/mL). Scale bars are 20 µm. B Ratio of mean fluorescent intensities of CD86:CD206 to mRFP1 fluorescent intensity as measured by confocal microscopy. C . Phenotyping via flow cytometry of RAW264.7-IRCs for CD86 (left) and CD206 (right) alongside mRFP1 expression under three conditions: unstained, non-stimulated, and LPS treated (10 ng/mL). The black arrows showcase the magnitude of the cell population shift due to LPS stimulation

Article Snippet: The mRFP1 red fluorescent protein was taken from the pU6-pegRNA-GG-acceptor plasmid (Addgene #132,777) via PCR using Q5 High-Fidelity DNA Polymerase (NEB) and purified.

Techniques: Immunostaining, Marker, Expressing, Confocal Microscopy, Flow Cytometry

Journal: BMC Methods

Article Title: A dual-fluorescence assay for gene delivery vehicle screening in macrophages with an inflammation-inducible reporter construct

doi: 10.1186/s44330-025-00030-x

Figure Lengend Snippet: Evaluation of Inflammatory Response and Delivery Efficiency of Viral and Non-Viral Delivery Vehicles using RAW264.7-IRCs. A Evaluation of viral and non-viral delivery methods using a dual-reporter assay at 24 and 48 h post-delivery. Efficiency of delivery and inflammatory response were determined by using different viral (AAV 1, 2, 5, 8, 9 and Ad5/35) and non-viral (TransIT-X2 and Lipofectamine 2000) delivery methods carrying a GFP payload to RAW264.7-IRCs at timepoints of 24 h and 48 h post-delivery. %GFP + representing efficient delivery and %mRFP1 + representing inflammatory response. Percentages represent the average of 3 biological replicates. Each bar represents the average percentages from 3 biological replicates. TransIT-X2 and Lipofectamine 2000 were only evaluated at 48 h post-transfection per manufacturer protocol. Raw data points can be found in Table S4. B Evaluation of the geometric mean of GFP to mRFP1 at 24 and 48 h post-delivery. The data represented here is the same samples in 3A. Individual data points were plotted for each biological replicate. Raw data points can be found in Table S4. Statistics done using One-Way Anova and Tukey’s Post-Hoc Analysis can be found in Table S5 for 24 h and S6 for 48 h. C Representative cell populations from dual-fluorescence reporter assay. Populations represented here are RAW264.7 cells with no reporter to set the negative control for GFP/mRFP1, non-treated RAW264.7-IRCS (NT) to determine basal mRFP1 expression among reporter cells, and then three representative populations from RAW264.7-IRCs treated with AAV 1, Ad 5/35, and TransIT-X2, respectively. Gating strategy for dual-fluorescence assay can be found in Figure S2

Article Snippet: The mRFP1 red fluorescent protein was taken from the pU6-pegRNA-GG-acceptor plasmid (Addgene #132,777) via PCR using Q5 High-Fidelity DNA Polymerase (NEB) and purified.

Techniques: Reporter Assay, Transfection, Fluorescence, Negative Control, Expressing

Journal: bioRxiv

Article Title: A Dual-Fluorescence Assay for Gene Delivery Vehicle Screening in Macrophages with an Inflammation-Inducible Reporter Construct

doi: 10.1101/2024.08.05.606664

Figure Lengend Snippet: 1A . Overview of RAW264.7-IRC cell line creation. The inflammatory reporter construct, created using Gibson cloning, contained an mCherry fluorescent reporter under an inflammatory promoter, termed synthesis friendly inflammatory promoter (SFNp) , and was encapsulated and transduced via lentivirus. The cells then underwent puromycin selection to create a homogenous population of cells. RAW264.7-IRCs were then stimulated using different cytokines and inflammatory agents to induce mCherry expression. 1B . LPS dose curve showing range of mCherry fluorescence geometric mean values measured using flow cytometry across a range of LPS doses from 0.001 to 100 ng/mL. Individual data points are plotted for each biological replicate. Each dose was done in triplicate. Trend line calculated using the three-parameter dose-response curve, which yielded an EC50 = 0.61 ng/mL with an R 2 value of 0.94. Dose-response curve line equation can be found in Table S1 . 1C . TNF-α concentration levels (pg/mL) in cell culture supernatant measured using ELISA. Cells were treated with LPS at concentrations ranging from 0.001 to 1 ng/mL for 24 hours. Each data point represents an average of 2 technical replicates. Trend line calculated using the three-parameter dose-response curve, which yielded an EC50 = 0.87 ng/mL with an R 2 value of 0.987. Dose-response curve line equation can be found in Table S2. 1D . Plot of mCherry fluorescent geometric mean values plotted against respective TNF-α concentration levels (pg/mL). X -values represent the average of the 3 biological replicates of TNF-α concentration (from ) and y -values represent the average of the 3 biological replicates of mCherry geometric mean (from ) ± SD. Trend line calculated using linear regression, y = 21.12 x -8156, with an R 2 value of 0.90. Linear regression parameters can be found in Table S3. 1E . RAW264.7-IRC responsiveness. RAW264.7-IRCs were treated with LPS and IFN-γ for 24 hours, 24h cells were harvested for flow cytometry. Stimulation media was replaced with fresh media for the 48h and 72h samples. 24h post-removal of stimulation media, the 48h samples were harvested for flow cytometry, and the same was done for the 72h samples. Mean ± SD, with individual data points for each biological replicate. (**** p-value ≤ 0.0001, *** p-value ≤ 0.001, ** p-value ≤ 0.01, n.s. p-value > 0.05). 1F . Timeline of treatment for RAW264.7-IRC responsiveness.

Article Snippet: The mCherry red fluorescent protein was taken from the pU6-pegRNA-GG-acceptor plasmid (Addgene #132777) via PCR using Q5 High-Fidelity DNA Polymerase (NEB) and purified.

Techniques: Construct, Cloning, Selection, Expressing, Fluorescence, Flow Cytometry, Concentration Assay, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: A Dual-Fluorescence Assay for Gene Delivery Vehicle Screening in Macrophages with an Inflammation-Inducible Reporter Construct

doi: 10.1101/2024.08.05.606664

Figure Lengend Snippet: 2A . Phenotyping via immunostaining of RAW264.7-IRCs for CD206 (green), a common anti-inflammatory marker, and CD86 (blue), a common inflammatory marker, co-localized with mCherry expression between no treatment and LPS treated cells (10ng/mL). Scale bars are 20 μm. 2B . Co-localization of CD86 and CD206 with mCherry expression in non-stimulated RAW264.7-IRCs. 2C . Co-localization of CD86 and CD206 with mCherry expression in LPS-stimulated RAW264.7-IRCs. 2D. Ratio of mean fluorescent intensities of CD86:CD206 to mCherry fluorescent intensity.

Article Snippet: The mCherry red fluorescent protein was taken from the pU6-pegRNA-GG-acceptor plasmid (Addgene #132777) via PCR using Q5 High-Fidelity DNA Polymerase (NEB) and purified.

Techniques: Immunostaining, Marker, Expressing

Journal: bioRxiv

Article Title: A Dual-Fluorescence Assay for Gene Delivery Vehicle Screening in Macrophages with an Inflammation-Inducible Reporter Construct

doi: 10.1101/2024.08.05.606664

Figure Lengend Snippet: 2A . Evaluation of viral and non-viral delivery methods using a dual-reporter assay at 24 and 48 hours post-delivery. Efficiency of delivery and inflammatory response were determined by using different viral (AAV 1, 2, 5, 8, 9 and Ad5/35) and non-viral (TransIT-X2 and Lipofectamine 2000) delivery methods carrying a GFP payload to RAW264.7-IRCs at timepoints of 24 hours and 48 hours post-delivery. %GFP+ representing efficient delivery and %mCherry+ representing inflammatory response. Percentages represent the average of 3 biological replicates. Each bar represents the average percentages from 3 biological replicates. TransIT-X2 and Lipofectamine 2000 were only evaluated at 48 hours post-transfection per manufacturer protocol. Raw data points can be found in Table S4. 2B . Evaluation of the geometric mean of GFP to mCherry at 24 and 48 hours post-delivery. The data represented here is the same samples in 2A. Individual data points were plotted for each biological replicate. Y-axes between 24 and 48 hours is different. Raw data points can be found in Table S4. Statistics done using One-Way Anova and Tukey’s Post-Hoc Analysis can be found in Table S5 for 24-hour and S6 for 48-hour. 2C . Representative cell populations from dual-fluorescence reporter assay. Populations represented here are RAW264.7 cells with no reporter to set the negative control for GFP/mCherry, non-treated RAW264.7-IRCS (NT) to determine basal mCherry expression among reporter cells, and then three representative populations from RAW264.7-IRCs treated with AAV 1, Ad 5/35, and TransIT-X2, respectively. Gating strategy for dual-fluorescence assay can be found in Figure S2.

Article Snippet: The mCherry red fluorescent protein was taken from the pU6-pegRNA-GG-acceptor plasmid (Addgene #132777) via PCR using Q5 High-Fidelity DNA Polymerase (NEB) and purified.

Techniques: Reporter Assay, Transfection, Fluorescence, Negative Control, Expressing

Journal: bioRxiv

Article Title: AI-based antibody discovery platform identifies novel, diverse and pharmacologically active therapeutic antibodies against multiple SARS-CoV-2 strains

doi: 10.1101/2023.08.21.554197

Figure Lengend Snippet: Unpurified transfection supernatants were tested by AlphaLISA for binding to biotinylated SARS-CoV-2 spike protein (red symbols) and in parallel to biotinylated irrelevant target protein (black symbols). Positive control antibody M-3418 (S309) demonstrates binding specificity, whereas isotype control antibody demonstrates lack of binding. Replicates were included when possible. Data was graphed using GraphPad Prism software. Representative binding profile shown.

Article Snippet: Subsequently, 10 μl of Protein A AlphaLISA ® Acceptor beads (10 μg/ml final concentration; Perkin Elmer cat# AL101C) were added, and further incubated for 1 hour at room temperature in the dark with shaking (250 rpm).

Techniques: Transfection, Binding Assay, Positive Control, Control, Software

Journal: bioRxiv

Article Title: AI-based antibody discovery platform identifies novel, diverse and pharmacologically active therapeutic antibodies against multiple SARS-CoV-2 strains

doi: 10.1101/2023.08.21.554197

Figure Lengend Snippet: Binding screen allows identification of target-specific binders. Unpurified transfection supernatants were tested by AlphaLISA for binding to biotinylated SARS-CoV-2 Spike protein (red symbols) and in parallel to biotinylated irrelevant target protein (black symbols). Positive control antibody S309 demonstrates binding specificity, whereas isotype control antibody demonstrates lack of binding. to Replicates were included when possible. Data was graphed using GraphPad Prism software. Representative binding profiles are shown.

Article Snippet: Subsequently, 10 μl of Protein A AlphaLISA ® Acceptor beads (10 μg/ml final concentration; Perkin Elmer cat# AL101C) were added, and further incubated for 1 hour at room temperature in the dark with shaking (250 rpm).

Techniques: Binding Assay, Transfection, Positive Control, Control, Software

Journal: bioRxiv

Article Title: AI-based antibody discovery platform identifies novel, diverse and pharmacologically active therapeutic antibodies against multiple SARS-CoV-2 strains

doi: 10.1101/2023.08.21.554197

Figure Lengend Snippet: Unpurified transfection supernatants were concentration normalized and tested for binding in a 9-point serial titration series to biotinylated SARS-CoV-2 Spike protein by AlphaLISA. Replicates were included when possible. Data was graphed using GraphPad Prism software. Representative binding profiles for 30 J.HAL ® antibodies are shown in the sub-plots A-F above, along with the positive control antibody M-3418 (S309).

Article Snippet: Subsequently, 10 μl of Protein A AlphaLISA ® Acceptor beads (10 μg/ml final concentration; Perkin Elmer cat# AL101C) were added, and further incubated for 1 hour at room temperature in the dark with shaking (250 rpm).

Techniques: Transfection, Concentration Assay, Binding Assay, Titration, Software, Positive Control

Journal: bioRxiv

Article Title: AI-based antibody discovery platform identifies novel, diverse and pharmacologically active therapeutic antibodies against multiple SARS-CoV-2 strains

doi: 10.1101/2023.08.21.554197

Figure Lengend Snippet: Unpurified transfection supernatants were concentration normalized and tested for binding in a 9-point serial titration series to biotinylated SARS-CoV-2 Spike protein by AlphaLISA. Replicates were included when possible. Data was graphed using GraphPad Prism software. Representative binding profiles for 43 J.HAL ® antibodies are shown in the sub-plots A-F above, along with the positive control antibody M-3418 (S309).

Article Snippet: Subsequently, 10 μl of Protein A AlphaLISA ® Acceptor beads (10 μg/ml final concentration; Perkin Elmer cat# AL101C) were added, and further incubated for 1 hour at room temperature in the dark with shaking (250 rpm).

Techniques: Transfection, Concentration Assay, Binding Assay, Titration, Software, Positive Control