fitc conjugated goat anti mouse  (Novus Biologicals)

Journal: Science Advances

Article Title: In vivo membrane engineering traps Gd-based MRI contrast agents for detecting microhepatocellular carcinoma

doi: 10.1126/sciadv.aec9913

Figure Lengend Snippet: ( A ) Representative Western blot analysis of GPC3 expression in HepG2, WRL-68, and Hepa1-6 cells ( n = 3 independent experiments). ( B ) Quantitation of relative GPC3 protein level from (A). Data are presented as means ± SD ( n = 3). Statistical analysis was performed using one-way ANOVA with a Tukey’s post hoc test. ( C ) Flow cytometry analysis of surface GPC3 expression in HepG2, WRL-68, and Hepa1-6 cells. ( D ) Representative IHC images of GPC3 expression (brown) in tumor tissues from orthotopic Hepa1-6 tumor-bearing mice. Scale bar, 50 μm. ( E ) CLSM images of HepG2 and Hepa1-6 cells treated with SPD1 or SPD2 nanoparticles (50 μM; red fluorescence) for 6 hours. Scale bars, 20 μm. ( F ) Time-dependent CLSM imaging of HepG2 cells treated with SPD1 nanoparticles (50 μM) showing membrane-localized fibrillar transformation. Scale bars, 20 μm. ( G ) CLSM analysis of HepG2 cells sequentially incubated with SPD1 or SPD2 nanoparticles (50 μM, 6 hours; red) and FITC-labeled anti-GPC3 antibody (green; 1:200; Abcam, #ab207080). Colocalization (yellow) indicates specific binding of SPD1 to membrane-bound GPC3. Fluorescence intensity and colocalization were quantified using MATLAB. Data are presented as means ± SD ( n = 3); n.s., not significant (one-way ANOVA with Tukey’s post hoc test). Scale bars, 20 μm. ( H ) SEM images of untreated HepG2 and WRL-68 cells or incubated with SPD1 or SPD2 nanoparticles (50 μM, 6 hours). Magnified insets highlight membrane-associated fibrillar structures. ( I ) TEM images of untreated HepG2 cells (top) and those treated with SPD1 nanoparticles (50 μM, 24 hours; bottom). Red arrows indicate membrane-associated nanofibers. Scale bars, 500 nm. ( J ) SEM images showing the persistence of SPD1-derived fibrillar networks on HepG2 cells at 6, 24, and 72 hours posttreatment (50 μM). Scale bars, 2 μm. All experiments were independently repeated three times with consistent and reproducible results.

Article Snippet: After three PBS washes (5 min each), cells were incubated with an FITC-conjugated goat anti-mouse IgG secondary antibody (1:1000; Abbkine, A23210) for 1 hour at room temperature in the dark.

Techniques: Western Blot, Expressing, Quantitation Assay, Flow Cytometry, Fluorescence, Imaging, Membrane, Transformation Assay, Incubation, Labeling, Binding Assay, Derivative Assay

Journal: Science Advances

Article Title: In vivo membrane engineering traps Gd-based MRI contrast agents for detecting microhepatocellular carcinoma

doi: 10.1126/sciadv.aec9913

Figure Lengend Snippet: ( A ) Representative CLSM images of HepG2 cells incubated with SPD1 nanoparticles (red, 50 μM) for 6 hours, followed by treatment with FITC-N 3 (10 to 50 μM, green) for an additional 6 hours. Merged yellow fluorescence indicates successful copper-free click conjugation between DBCO and N 3 on the cell membrane. Cells treated with SPD2+FITC-N 3 (50 μM) served as the nontargeted controls, showing minimal colocalization. Scale bars, 20 μm. ( B ) Time-dependent kinetics of bioorthogonal conjugation quantified by BCA protein assay and ICP-MS. HepG2 cells were pretreated with SPD1 or SPD2 nanoparticles (50 μM, 6 hours), followed by incubation with Gd-DOTA-N 3 (50 μM) for 0.5, 1, 6, or 12 hours. Cells treated with Gd-DOTA-N 3 alone served as baseline controls. ( C ) T 1 -weighted MR images of HepG2 cells treated with Gd-DOTA (50 μM), Gd-DOTA-N 3 (50 μM), or sequentially with SPD1 or SPD2 (50 μM, 6 hours) followed by Gd-DOTA-N 3 (50 μM, 6 hours). ( D ) Quantitative r 1 relaxivity under the corresponding treatment conditions in (C). ( E ) r 1 relaxivity of HepG2 cells preblocked with anti-GPC3 antibody (5 μg/ml, 12 hours; Abcam, #ab207080) before SPD1 treatment (50 μM, 6 hours) followed by Gd-DOTA-N 3 (50 μM, 6 hours). ( F to H ) Cell viability of WRL-68 (F), HepG2 (G), and Hepa1-6 (H) cells after sequential treatment with SPD1 or SPD2 for 6 hours followed by Gd-DOTA-N 3 (50 μM, 6 hours). Cell viability was quantified using the CCK-8 assay. Data are presented as means ± SD { n = 3 for [(A) to (E)]; n = 6 for [(F) to (H)]}. Statistical significance was performed using one-way ANOVA followed by Tukey’s post hoc test. P < 0.05 was considered statistically significant; n.s., not significant. All experiments were independently repeated three times with consistent results.

Article Snippet: After three PBS washes (5 min each), cells were incubated with an FITC-conjugated goat anti-mouse IgG secondary antibody (1:1000; Abbkine, A23210) for 1 hour at room temperature in the dark.

Techniques: Incubation, Fluorescence, Conjugation Assay, Membrane, Bicinchoninic Acid Protein Assay, CCK-8 Assay