Journal: Frontiers in Drug Discovery

Article Title: Mimicking the immunosuppressive impact of fibroblasts in a 3D multicellular spheroid model

doi: 10.3389/fddsv.2024.1427407

Figure Lengend Snippet: FIGURE 5 Increase in FAP surface expression in spheroids compared to monolayer culture. For flow cytometry, cells were stained with two dilutions of PE- conjugated FAP antibody and SYTOX™Red dead cell stain. Measurements with PBS/1% BSA served as a negative control. (A) Surface antibody binding in 2D and 3D mono-culture of MDA-MB-231 fluc GFP cells, mono-culture of WS1 cells, and co-culture of both cell lines after 72 h. Only the viable cells were examined for the event analysis. The cells were separated into GFP-positive (GFP+) and GFP-negative (GFP-) populations. (B) The % fluorescence signal of the median of viable GFP- cells was calculated by normalizing to the respective negative controls. Statistical significance was calculated using a Kruskal–Wallis one-way ANOVA in GraphPad Prism v9.1.2; *p < 0.05; ***p < 0.0005. (mean ± SEM, n = 3; n = 2 2D mono-culture WS1 1:50 AB).

Article Snippet: Thereafter, cells were washed twice with cold 1x DPBS supplemented with 1% BSA and stained with a PE-labeled antihuman FAPα antibody (Cat. No. FAB3715P, R&D Systems) or with a TAMRA-dextran-oncoFAP construct for 30 min on ice in the dark.

Techniques: Expressing, Cytometry, Staining, Negative Control, Binding Assay, Co-Culture Assay

Journal: Frontiers in Drug Discovery

Article Title: Mimicking the immunosuppressive impact of fibroblasts in a 3D multicellular spheroid model

doi: 10.3389/fddsv.2024.1427407

Figure Lengend Snippet: FIGURE 6 Strong IFNγ release after simultaneous FAP and CD3 binding through oncoFAP-bearing construct. (A) Labeling of alkyne-dextran-cadaverine with NHS-TAMRA and subsequent attachment of multiple oncoFAP moieties via CuAAC. Created with BioRender.com. (B) The % fluorescence signal was calculated by normalizing the median of viable GFP- cells stained with TAMRA-dextran-oncoFAP to the median viable GFP- unstained cells measured by flow cytometry. The graph was created in GraphPad Prism v9.1.2. (mean ± SEM, n = 2). (C) mTG mediated conjugation of α-CD3-scFv-Fc protein and alkyne-dex-cadaverine. Subsequently, oncoFAP was introduced via CuAAC. Created with BioRender.com. (D) The α-CD3-scFv-Fc-dextran-oncoFAP construct (abbreviated as construct) was added together with naïve T cells on day 2. As control α-CD3-scFv-Fc (abbreviated as CD3) alone was tested. IFNγ concentration [pg/mL] was measured in triple-culture after 48 h of treatment. The dotted line represents the lowest standard. Concentrations were calculated with GraphPad Prism v9.1.2 and statistical significance was calculated using a Kruskal–Wallis one-way ANOVA in GraphPad Prism v9.1.2; ***p < 0.001. (mean ± SEM; n = 3 with 8 different T cell donors for α-CD3-scFv-Fc-Dex-oncoFAP, n = 1 with 3 different T cell donors for α-CD3-scFv-Fc).

Article Snippet: Thereafter, cells were washed twice with cold 1x DPBS supplemented with 1% BSA and stained with a PE-labeled antihuman FAPα antibody (Cat. No. FAB3715P, R&D Systems) or with a TAMRA-dextran-oncoFAP construct for 30 min on ice in the dark.

Techniques: Binding Assay, Construct, Labeling, Staining, Cytometry, Conjugation Assay, Control, Concentration Assay