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(A) Supernatants from HCoV-229E-infected human cell lines (MRC-5, Huh7.5, and <t>HCT-8;</t> top to bottom rows) were stained using S-specific mAbs targeting the RBD (C04), S1 (F07), or S2 (F12) domains, or an isotype control (IgG), followed by a PE-labeled secondary antibody. Gates for positive staining were set above the isotype control background (>15 PE MESF on the y-axis). Calibrated mean PE fluorescence values (MESF) from gated regions are shown in red. (B) Quantitative comparison of staining results from the gates in (A), with bars representing mean PE MESF ± standard deviation wherein light blue = MRC-5, blue = Huh7.5, and dark blue = HCT-8. Data are representative of at least three independent replicates. Statistical significance was determined using two-way ANOVA with Dunnett’s multiple comparisons test to compare each antibody condition to the IgG control within each virus. Significance thresholds: **p < 0.01, ***p < 0.001, ****p < 0.0001.(C) HCoV-229E pseudoviruses produced by transfection of 293T cells were stained directly in cell culture supernatants using the same antibody panel and staining protocol as in (A). (D) Quantification of staining in (C), with bars indicating mean PE MESF ± standard deviation. See also Figures S1 - S3.
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(A) Supernatants from HCoV-229E-infected human cell lines (MRC-5, Huh7.5, and <t>HCT-8;</t> top to bottom rows) were stained using S-specific mAbs targeting the RBD (C04), S1 (F07), or S2 (F12) domains, or an isotype control (IgG), followed by a PE-labeled secondary antibody. Gates for positive staining were set above the isotype control background (>15 PE MESF on the y-axis). Calibrated mean PE fluorescence values (MESF) from gated regions are shown in red. (B) Quantitative comparison of staining results from the gates in (A), with bars representing mean PE MESF ± standard deviation wherein light blue = MRC-5, blue = Huh7.5, and dark blue = HCT-8. Data are representative of at least three independent replicates. Statistical significance was determined using two-way ANOVA with Dunnett’s multiple comparisons test to compare each antibody condition to the IgG control within each virus. Significance thresholds: **p < 0.01, ***p < 0.001, ****p < 0.0001.(C) HCoV-229E pseudoviruses produced by transfection of 293T cells were stained directly in cell culture supernatants using the same antibody panel and staining protocol as in (A). (D) Quantification of staining in (C), with bars indicating mean PE MESF ± standard deviation. See also Figures S1 - S3.
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(A) Supernatants from HCoV-229E-infected human cell lines (MRC-5, Huh7.5, and HCT-8; top to bottom rows) were stained using S-specific mAbs targeting the RBD (C04), S1 (F07), or S2 (F12) domains, or an isotype control (IgG), followed by a PE-labeled secondary antibody. Gates for positive staining were set above the isotype control background (>15 PE MESF on the y-axis). Calibrated mean PE fluorescence values (MESF) from gated regions are shown in red. (B) Quantitative comparison of staining results from the gates in (A), with bars representing mean PE MESF ± standard deviation wherein light blue = MRC-5, blue = Huh7.5, and dark blue = HCT-8. Data are representative of at least three independent replicates. Statistical significance was determined using two-way ANOVA with Dunnett’s multiple comparisons test to compare each antibody condition to the IgG control within each virus. Significance thresholds: **p < 0.01, ***p < 0.001, ****p < 0.0001.(C) HCoV-229E pseudoviruses produced by transfection of 293T cells were stained directly in cell culture supernatants using the same antibody panel and staining protocol as in (A). (D) Quantification of staining in (C), with bars indicating mean PE MESF ± standard deviation. See also Figures S1 - S3.

Journal: bioRxiv

Article Title: Evaluating Spike Antigenicity across Endemic Human Coronavirus Models using Flow Virometry

doi: 10.64898/2026.05.28.728498

Figure Lengend Snippet: (A) Supernatants from HCoV-229E-infected human cell lines (MRC-5, Huh7.5, and HCT-8; top to bottom rows) were stained using S-specific mAbs targeting the RBD (C04), S1 (F07), or S2 (F12) domains, or an isotype control (IgG), followed by a PE-labeled secondary antibody. Gates for positive staining were set above the isotype control background (>15 PE MESF on the y-axis). Calibrated mean PE fluorescence values (MESF) from gated regions are shown in red. (B) Quantitative comparison of staining results from the gates in (A), with bars representing mean PE MESF ± standard deviation wherein light blue = MRC-5, blue = Huh7.5, and dark blue = HCT-8. Data are representative of at least three independent replicates. Statistical significance was determined using two-way ANOVA with Dunnett’s multiple comparisons test to compare each antibody condition to the IgG control within each virus. Significance thresholds: **p < 0.01, ***p < 0.001, ****p < 0.0001.(C) HCoV-229E pseudoviruses produced by transfection of 293T cells were stained directly in cell culture supernatants using the same antibody panel and staining protocol as in (A). (D) Quantification of staining in (C), with bars indicating mean PE MESF ± standard deviation. See also Figures S1 - S3.

Article Snippet: HCT-8 (HRT-18) cells (ATCC, Cat# CCL-244) were cultured in RPMI (Gibco, Cat#11875-093).

Techniques: Infection, Staining, Control, Labeling, Fluorescence, Comparison, Standard Deviation, Virus, Produced, Transfection, Cell Culture