7aad staining solution  (Miltenyi Biotec)

Journal: Vaccines

Article Title: Modified Hematopoietic Stem Cell-Derived Dendritic Cell Therapy Retained Tumor-Inhibitory Function and Led to Regression of Primary and Metastatic Pancreatic Tumors in Humanized Mouse Models

doi: 10.3390/vaccines13111131

Figure Lengend Snippet: LVV Optimization. ( A ) Flow cytometry results of feasibility study testing the effect of varying MOIs (5, 10, and 20) and promoter strength (EFS vs. EF1α) on MFI of CD93 and CD40L expression illustrated in raw MFI, difference in MFI between the EFS and EF1α viruses at indicated MOI, and fold change, n = 4. EFS virus results are identified as blue points whereas EF1α virus results are identified as red points. ( B ) MFI results of CD93 and CD40L with EFS (EFS_CD93_CD40L_CXCL13) and six EF1α viruses (all possible combinations of transgene orientations) illustrated as MFI mean ± SD on the left and MFI fold changes relative to the EFS control virus on the right graph, n = 4, MOI = 5. EFS virus is identified by the red dashed box on left graph and results are normalized to said group on the right graph by a red dashed line when compared by fold change. ( C ) Percentage of 7AAD-positive cells corresponding to each experimental condition on left graph, n = 4. Lentivirus yield from two rounds of large-scale packaging on right graph, n = 4. ( D ) CXCL13 and CD40L protein concentration in cell supernatant determined by ELISA, n = 2. Protein concentration fold changes relative to the virus with EFS promoter. ( E ) Vector maps illustrating 1st Gen EFS control and 2nd Gen LVV that were selected for use in future in vitro and in vivo experiments. Star annotations highlight modifications to vector construct, such as EF1α promoter from EFS, new transgene orientation with CD40L in 1st ORF, CD93 in 2nd ORF, and CXCL13 in 3rd ORF, oPRE operator from WPRE, and kanamycin antibiotic resistance from ampicillin. “(ns)” stands for nonsynonymous describing the type of gene variant.

Article Snippet: Propidium iodide or 7AAD (Miltenyi Biotec) was used to evaluate cell viability.

Techniques: Flow Cytometry, Expressing, Virus, Control, Protein Concentration, Enzyme-linked Immunosorbent Assay, Plasmid Preparation, In Vitro, In Vivo, Construct, Variant Assay

Journal: Frontiers in Immunology

Article Title: Itolizumab regulates activating and inhibitory signals on effector cells, improving their cytotoxicity against CD318+ tumor cell lines

doi: 10.3389/fimmu.2025.1585597

Figure Lengend Snippet: Itolizumab enhanced tumor cell killing by PBMC challenged with CD318+ tumor cell lines. PBMC were maintained untreated (UT, black dots) or were pre-incubated with 10 µg/mL of isotype control (IC, green dots) or itolizumab (T1h, blue dots) and challenged with (A) MDA-MB-231 (n=10), (B) NCI-H460 (n=10), (C) SKOV-3 (n=6), (D) HCT-116 (n=6) and (E) MCF-7 (n=10) human tumor cell lines. Tumor cell lysis was measured using 7AAD staining by flow cytometry. Representative dot plots for each condition and percentage of tumor cell viability in the co-cultures for each donor are shown. Data are depicted as median ± 95% confidence interval. Statistical analysis was performed using one-way ANOVA and Tukey’s multiple comparisons test, or Kruskal-Wallis test and Dunn’s multiple comparisons test, both for unpaired data. Only statistical significance is shown in the graphs, with *p ≤ 0.05, and ****p ≤ 0.0001.

Article Snippet: 7-Aminoactinomycin D (7AAD) staining solution (130-111-568, Miltenyi Biotec, USA) was used as a cell death marker.

Techniques: Incubation, Control, Lysis, Staining, Flow Cytometry

Journal: Frontiers in Immunology

Article Title: Itolizumab regulates activating and inhibitory signals on effector cells, improving their cytotoxicity against CD318+ tumor cell lines

doi: 10.3389/fimmu.2025.1585597

Figure Lengend Snippet: Itolizumab increased the tumor cell killing capacity of PBMC by blocking the inhibitory effects associated with CD6-CD318 interaction. (A) PBMC and MDA-MB-231, NCI-H460, SKOV-3 and HCT-116 cell lines (n=4) were pre-incubated with 10 µg/mL of isotype control (IC, green), itolizumab (T1h, blue), or neutralizing antibodies specific for CD318 (brown) or ALCAM (yellow). Effector and target cells were then co-cultured, and tumor cell lysis was measured using 7AAD staining by flow cytometry. (B) CFSE-labeled T-cells (n=6) were activated with antiCD3/CD28 beads and incubated with 10ug/mL of pre-coated human recombinant CD318 (brown), ALCAM (yellow), or PBS (green) as control. (C) Immune cell viability was measured on isotype control treated-PBMC co-cultured with human tumor cell lines MDA-MB-231 (n=10, red) and MCF-7 (n=10, green) using flow cytometry. Representative histograms or dot plots for each condition, and individual viability percentage and CFSE dilution are displayed. Data are depicted as median ± 95% confidence interval. Statistical analysis was performed using the Kruskal-Wallis test and Dunn’s multiple comparisons test with unpaired data. Only statistical significance is shown in the graphs, with *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001.

Article Snippet: 7-Aminoactinomycin D (7AAD) staining solution (130-111-568, Miltenyi Biotec, USA) was used as a cell death marker.

Techniques: Blocking Assay, Incubation, Control, Cell Culture, Lysis, Staining, Flow Cytometry, Labeling, Recombinant

Journal: Frontiers in Immunology

Article Title: Itolizumab regulates activating and inhibitory signals on effector cells, improving their cytotoxicity against CD318+ tumor cell lines

doi: 10.3389/fimmu.2025.1585597

Figure Lengend Snippet: Itolizumab enhanced tumor cell killing by activating CD8+ and NK cells challenged with CD318+ tumor cell lines. PBMC and purified CD4+ and CD8+ T-cells and NK cells were maintained untreated (UT, black) or were pre-incubated with 10 µg/mL of isotype control (IC, green) or itolizumab (T1h, blue) and challenged with MDA-MB-231 (n=6). (A) Tumor cell viability was measured using 7AAD staining by flow cytometry. The frequency of NKG2D+, CD69+, and granzyme B+ in (B, D ) CD8+ T and (C, E ) NK cells in the co-cultures were determined by flow cytometry. Representative dot plots or histograms of each condition and individual percentage of positive cells are displayed. IFNγ levels on co-culture supernatants of MDA-MB-231 with enriched CD8+ T (F) and NK cells (G) were quantified by ELISA. Individual values of IFNγ concentration (pg/mL) per donor are shown. Data are depicted as median ± 95% confidence interval. Statistical analysis was performed using one-way ANOVA and Tukey’s multiple comparisons test. Only statistical significance is shown in the graphs, with ***p ≤ 0.001, and ****p ≤ 0.0001.

Article Snippet: 7-Aminoactinomycin D (7AAD) staining solution (130-111-568, Miltenyi Biotec, USA) was used as a cell death marker.

Techniques: Purification, Incubation, Control, Staining, Flow Cytometry, Co-Culture Assay, Enzyme-linked Immunosorbent Assay, Concentration Assay

Journal: Frontiers in Immunology

Article Title: Itolizumab regulates activating and inhibitory signals on effector cells, improving their cytotoxicity against CD318+ tumor cell lines

doi: 10.3389/fimmu.2025.1585597

Figure Lengend Snippet: Increase of PD-1 and CTL-4 expression on CD8+ T cells by itolizumab promotes an additive cytotoxic effect in PBMC treated with combinations of itolizumab and other ICI. Isolated CD8+ T cells and NK cells (n=6) from healthy controls were maintained untreated (UT, black) or pre-incubated with 10 µg/mL isotype control (IC, green) or itolizumab (T1h, blue) and challenged with breast tumor cell line MDA-MB-231. Representative dot plots and frequency of PD1+ and CTLA4+ of (A) NK and (B) CD8+ T cells per donor are shown. (C) PBMC from healthy individuals were maintained untreated (UT, black dots) or pre-incubated with 10 µg/mL isotype control (IC, green dots), pembrolizumab (P, black dots), itolizumab (T1h, blue dots) or a combination of both (T1h+P, bicolor dots) and challenged with CD318+ breast tumor cell line MDA-MB-231. Tumor cell viability was measured by 7AAD staining by flow cytometry. Representative dot plots of 7AAD staining for each treatment and individual tumor cell viability percentages for each condition are displayed. Data are depicted as median ± 95% confidence interval. Statistical analysis was performed using one-way ANOVA and Tukey’s multiple comparisons test. Only statistical significance is shown in the graphs, with **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001.

Article Snippet: 7-Aminoactinomycin D (7AAD) staining solution (130-111-568, Miltenyi Biotec, USA) was used as a cell death marker.

Techniques: Expressing, Isolation, Incubation, Control, Staining, Flow Cytometry