Journal: Neuro-Oncology

Article Title: Agonist OX40 immunotherapy improves survival in glioma-bearing mice and is complementary with vaccination with irradiated GM-CSF–expressing tumor cells

doi: 10.1093/neuonc/nox125

Figure Lengend Snippet: Th1 vs Th2 BILs. (A) Flow cytometry demonstrating percent expression of T-bet, IFN-γ, and IL-12R β2 on CD4+ lymphocytes 18 days after GL261 tumor implantation by treatment group. Combination immunotherapy is associated with significant increases in all 3 Th1 markers. (***P < 0.0005, **P < 0.005, *P < 0.05; absence of an asterisk denotes not significant comparison.) (B) Flow cytometry demonstrating percent expression of GATA3, the IL-4 receptor, and IL-5 on CD4+ lymphocytes 18 days after GL261 tumor implantation by treatment group. Combination immunotherapy is associated with significant decreases in all 3 Th2 markers. (**P < 0.005, *P < 0.05, absence of an asterisk denotes not significant comparison.)

Article Snippet: For the analysis of T helper cell subsets, we used Multi-Color Flow Cytometry Kits (R&D Systems) for mouse Th1 cells (conjugated antibodies to T-bet-PerCP, IFN-gamma-fluorescein, IL-12 R beta 2-APC, and CD4-PE) and Th2 cells (conjugated antibodies to CD4-PerCP, IL-4 R-fluorescein, STAT6-APC, and IL-5-PE).

Techniques: Flow Cytometry, Expressing, Tumor Implantation, Comparison

Journal: Neuro-Oncology

Article Title: Agonist OX40 immunotherapy improves survival in glioma-bearing mice and is complementary with vaccination with irradiated GM-CSF–expressing tumor cells

doi: 10.1093/neuonc/nox125

Figure Lengend Snippet: Day 18 flow cytometry of BILs shows that vaccination improves the intratumoral CD8+/FoxP3+ lymphocyte ratio, which is little affected by treatment with anti-OX40 immunotherapy. (**P < 0.005, *P < 0.05; absence of an asterisk denotes not significant comparison.)

Article Snippet: For the analysis of T helper cell subsets, we used Multi-Color Flow Cytometry Kits (R&D Systems) for mouse Th1 cells (conjugated antibodies to T-bet-PerCP, IFN-gamma-fluorescein, IL-12 R beta 2-APC, and CD4-PE) and Th2 cells (conjugated antibodies to CD4-PerCP, IL-4 R-fluorescein, STAT6-APC, and IL-5-PE).

Techniques: Flow Cytometry, Comparison

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Alternating 2D and 3D culture reduces cell size and extends the lifespan of placenta-derived mesenchymal stem cells

doi: 10.3389/fbioe.2025.1632810

Figure Lengend Snippet: Isolation of Placenta-Derived MSCs. (A) Illustration of the MSC isolation process. (B) Representative morphology of MSCs during isolation. (C) Differentiation of MSCs into adipocytes (FABP-4 + ) and osteocytes (osteocalcin + ). (D) Flow cytometry analysis of MSC surface markers. Negative markers include CD34, CD45, CD11b, CD79A, and HLA-DR. (E) Size distribution of MSCs cultured in T-25 flasks at passages 4 (P4), 6 (P6), and 8 (P8).

Article Snippet: MSCs were characterized using the Human Mesenchymal Stem Cell Verification Flow Kit (R&D Systems), which includes antibodies against the positive markers CD90, CD73, and CD105, as well as the negative markers CD45, CD34, CD11b, CD79A, and HLA-DR. Additionally, the Human Mesenchymal Stem Cells Multi-Color Flow Kit (R&D Systems) was used to assess expression of the positive markers CD44, CD106, CD146, and CD166.

Techniques: Isolation, Derivative Assay, Flow Cytometry, Cell Culture

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Alternating 2D and 3D culture reduces cell size and extends the lifespan of placenta-derived mesenchymal stem cells

doi: 10.3389/fbioe.2025.1632810

Figure Lengend Snippet: 3D Spheroid Culture Reduces MSC Size: Identifying the Optimal Spheroid Diameter. (A) Representative images of MSC (P3) spheroids cultured for 3 days with varying initial cell numbers per spheroid. (B) Quantification of spheroid diameter over the 3-day culture period. (C) Representative images of individual MSCs following 2D and 3D spheroid culture. (D,E) Cell size distribution of MSCs after 2D and 3D spheroid culture. (F) Proportions of small (≤15 µm) and large (>15 µm) MSCs after 2D and 3D spheroid culture. Spheroid culture duration in (C–F) was 72 h.

Article Snippet: MSCs were characterized using the Human Mesenchymal Stem Cell Verification Flow Kit (R&D Systems), which includes antibodies against the positive markers CD90, CD73, and CD105, as well as the negative markers CD45, CD34, CD11b, CD79A, and HLA-DR. Additionally, the Human Mesenchymal Stem Cells Multi-Color Flow Kit (R&D Systems) was used to assess expression of the positive markers CD44, CD106, CD146, and CD166.

Techniques: Cell Culture

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Alternating 2D and 3D culture reduces cell size and extends the lifespan of placenta-derived mesenchymal stem cells

doi: 10.3389/fbioe.2025.1632810

Figure Lengend Snippet: 3D Spheroid Culture Reduces MSC Size: Determining the Optimal Culture Duration. (A) Representative images showing morphological changes in MSC (P4) spheroids over a 7-day culture period. (B) Images of individual MSCs cultured in 2D or in 25 K-cell spheroids for varying durations. (C) Changes in the diameter of 25 K-cell MSC 3D spheroids over 7 days. (D) Comparison of mean MSC diameter in 2D versus 25 K spheroid cultures over 7 days (E,F) Size distribution of MSCs within 25 K spheroids across the 7-day culture period.

Article Snippet: MSCs were characterized using the Human Mesenchymal Stem Cell Verification Flow Kit (R&D Systems), which includes antibodies against the positive markers CD90, CD73, and CD105, as well as the negative markers CD45, CD34, CD11b, CD79A, and HLA-DR. Additionally, the Human Mesenchymal Stem Cells Multi-Color Flow Kit (R&D Systems) was used to assess expression of the positive markers CD44, CD106, CD146, and CD166.

Techniques: Cell Culture, Comparison

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Alternating 2D and 3D culture reduces cell size and extends the lifespan of placenta-derived mesenchymal stem cells

doi: 10.3389/fbioe.2025.1632810

Figure Lengend Snippet: Effect of Chemically Defined Medium on MSC Viability and Size in Spheroid Culture. (A) Phase-contrast and dead cell staining (red) images of MSC (P6) spheroids cultured in various media. Three representative spheroids are shown in each condition. (B) MSC size distribution in 3D cultures across different media conditions. (C,D) Anti-inflammatory capability of MSCs cultured under varying conditions. RAW-Dual™ reporter cells were stimulated with 100 ng/mL LPS and 10 ng/mL IFNγ, then treated with MSCs. Dexamethasone (Dex, 1 µg/mL) was used as a positive control. Luciferase activity (C) and mouse IL-6 (D) were measured. Spheroid culture time was 48 h.

Article Snippet: MSCs were characterized using the Human Mesenchymal Stem Cell Verification Flow Kit (R&D Systems), which includes antibodies against the positive markers CD90, CD73, and CD105, as well as the negative markers CD45, CD34, CD11b, CD79A, and HLA-DR. Additionally, the Human Mesenchymal Stem Cells Multi-Color Flow Kit (R&D Systems) was used to assess expression of the positive markers CD44, CD106, CD146, and CD166.

Techniques: Staining, Cell Culture, Positive Control, Luciferase, Activity Assay

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Alternating 2D and 3D culture reduces cell size and extends the lifespan of placenta-derived mesenchymal stem cells

doi: 10.3389/fbioe.2025.1632810

Figure Lengend Snippet: Effects of Alternating 2D/3D Culture on MSC Size and Immunomodulatory Function. (A) MSCs were cultured in flasks for four passages, with an additional 2-day spheroid culture following each passage. Shown are the mean cell diameters immediately after 2D culture and after subsequent spheroid culture. (B) MSC diameters from P5 to P8 using conventional 2D culture versus the alternating 2D/3D method. P4 cells served as the starting population for both conditions. Diameters were measured after harvesting from 2D flasks. (C) Comparison of MSC sizes at P5–P8 between the two culture methods. (D) Comparison of doubling times at P5–P8 between the two methods. (E,F) Macrophages were activated with LPS and IFNγ, then treated with either early-passage (P4) or late-passage (P15) MSCs derived from conventional 2D culture or the alternating 2D/3D protocol. Levels of mouse IL-6 and IL-10 were measured to assess immunomodulatory effects.

Article Snippet: MSCs were characterized using the Human Mesenchymal Stem Cell Verification Flow Kit (R&D Systems), which includes antibodies against the positive markers CD90, CD73, and CD105, as well as the negative markers CD45, CD34, CD11b, CD79A, and HLA-DR. Additionally, the Human Mesenchymal Stem Cells Multi-Color Flow Kit (R&D Systems) was used to assess expression of the positive markers CD44, CD106, CD146, and CD166.

Techniques: Cell Culture, Comparison, Derivative Assay

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Alternating 2D and 3D culture reduces cell size and extends the lifespan of placenta-derived mesenchymal stem cells

doi: 10.3389/fbioe.2025.1632810

Figure Lengend Snippet: RGD-Modified Alginate Hydrogel Tubes (AlgTubes) for MSC Culture. (A) Schematic of alginate modification with RGD peptides. (B,C) Chemistry underlying hydrogel tube formation. (D,E) Process AlgTubes using a micro-extruder: a cell suspension and alginate solution are pumped into the central and side channels, respectively, creating coaxial core-shell flows. These are extruded through a nozzle into a CaCl 2 buffer, where Ca 2+ ions crosslink the outer alginate shell, forming hydrogel tubes instantly. (F) Illustration of growing MSCs within an AlgTube. (G,H) SEM images showing the porous structure of the AlgTubes.

Article Snippet: MSCs were characterized using the Human Mesenchymal Stem Cell Verification Flow Kit (R&D Systems), which includes antibodies against the positive markers CD90, CD73, and CD105, as well as the negative markers CD45, CD34, CD11b, CD79A, and HLA-DR. Additionally, the Human Mesenchymal Stem Cells Multi-Color Flow Kit (R&D Systems) was used to assess expression of the positive markers CD44, CD106, CD146, and CD166.

Techniques: Modification, Suspension

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Alternating 2D and 3D culture reduces cell size and extends the lifespan of placenta-derived mesenchymal stem cells

doi: 10.3389/fbioe.2025.1632810

Figure Lengend Snippet: Dynamic Cell Adhesion in RGD-Modified AlgTubes. (A) MSCs were processed into RGD-modified AlgTubes. (B–D) Cells adhered to the inner surface and proliferated from day 0 to day 6. (E) On day 6, free RGD peptides were added to the culture medium, leading to cell contraction within 24 h. (F) By 48 h, cells had fully detached and formed spheroids (red arrows). (G,H) Removal of free RGD peptides from the medium resulted in MSC reattachment to the AlgTube surface and resumed cell growth (blue arrows). (I–L) As a comparison, in the absence of free RGD peptides, day 6 MSCs continued to grow and eventually covered the inner surface of the hydrogel tube.

Article Snippet: MSCs were characterized using the Human Mesenchymal Stem Cell Verification Flow Kit (R&D Systems), which includes antibodies against the positive markers CD90, CD73, and CD105, as well as the negative markers CD45, CD34, CD11b, CD79A, and HLA-DR. Additionally, the Human Mesenchymal Stem Cells Multi-Color Flow Kit (R&D Systems) was used to assess expression of the positive markers CD44, CD106, CD146, and CD166.

Techniques: Modification, Comparison

Journal: Cancers

Article Title: Define Critical Parameters of Trastuzumab-Mediated ADCC Assays via Assay Optimization Processes, Focusing on the Impact of Cryopreserved Effector Cells on Assay Performance.

doi: 10.3390/cancers16132367

Figure Lengend Snippet: Figure 1. Influence of effector cell preparation, choice of effector cell, and E/T ratio on the measure- ment of trastuzumab-mediated ADCC using LDH ADCC assay. a–d. Influence of cryopreservation and the recovery cultivation time (4 h to overnight in RPMI-1640 with 10% FBS) on the recovery rate (a), viability of recovered PBMCs (b), the rate of NK cell isolation from the PBMCs (c), and the viability of isolated NK cells (d). Data were analyzed with GraphPad Prism using one-way ANOVA mixed-effects statistical analysis based on matched rows and recovery time as repeated measure with Tukey’s post hoc test. Overall p values for Figure 1a–d are <0.0001, 0.0126, 0.2553 (no statistical significance among three groups), and 0.0185, respectively; * p < 0.05 and **** p < 0.0001 (e) ADCC activity of trastuzumab with NK cells from immediately thawed PBMCs or overnight recovered PBMCs as effector cells with E/T ratio 20:1 measured after treatment for 5 h, 24 h, or 48 h. (f) ADCC activity of trastuzumab with immediately thawed or overnight recovered PBMCs as effector cells with E/T ratio 20:1 after treatment for 24 h. Data were analyzed with GraphPad Prism using two-way ANOVA with Šídák’s multiple comparisons test. Overall p value < 0.0001 for unrecovered compared to overnight recovered PBMCs. For (e,f), SKBR-3 cells were treated with trastuzumab (0–1 µg/mL) and NK cells, and killing of SKBR-3 was measured using the LDH ADCC assay. NC stands for the no-cell or media-only control, E stands for the effector-cell-only control, and T stands for the target-cell-only control. Data were analyzed with GraphPad Prism using two-way ANOVA, and p values are <0.0001. (g) Comparing Emax and EC50 values of LDH ADCC activity between different

Article Snippet: A FlowX Human NK Cell Phenotyping Flow Cytometry Kit (R&D Systems, Cat# FMC033, Minneapolis, MN, USA) was performed to assess NK cell phenotype using flow cytometry.

Techniques: ADCC Assay, Cell Isolation, Isolation, Activity Assay, Control

Journal: Cancers

Article Title: Define Critical Parameters of Trastuzumab-Mediated ADCC Assays via Assay Optimization Processes, Focusing on the Impact of Cryopreserved Effector Cells on Assay Performance.

doi: 10.3390/cancers16132367

Figure Lengend Snippet: Figure 2. Influence of the recovery culturing time and method on LDH ADCC assay and cellular expression of granulation proteins. (a) Comparison of ADCC activity of trastuzumab with NK cells isolated from PBMCs recovered for different times 0 h (NR), 4 h, or 24 h. Data were analyzed with GraphPad Prism using two-way ANOVA with Tukey’s multiple comparisons test. **** p < 0.0001; (b) Comparison of trastuzumab induced ADCC activity using NK cells that underwent overnight recovery after isolation from thawed cryopreserved PBMCs (R-iso for “recovered after isolation”), and NK cells isolated from the overnight recovered PBMCs (R-PB). Data were analyzed with GraphPad Prism using two-way ANOVA with Tukey’s multiple comparisons test. **** p < 0.0001. SKBR-3 cells were treated with trastuzumab (0–0.01 µg/mL) and NK cells with E/T ratio 5:1 for 24 h, and killing of SKBR-3 was measured with LDH ADCC assay; (c) Representative antiperforin, antigranzyme B, and Anti GAPDH immunoblot in NK cells isolated from unrecovered (NR) PBMCs or from PBMCs after recovery cultivation for different time points (4 h, 24 h) with E/T ratio 5:1. Lanes 1–3, 4–6, and 7–9 show expression levels for NK cells after incubation for 24 h hours with media only, with SKBR-3 only, and with SKBR-3 and trastuzumab, respectively. All lanes were run in the same gel, and images were

Article Snippet: A FlowX Human NK Cell Phenotyping Flow Cytometry Kit (R&D Systems, Cat# FMC033, Minneapolis, MN, USA) was performed to assess NK cell phenotype using flow cytometry.

Techniques: ADCC Assay, Expressing, Comparison, Activity Assay, Isolation, Western Blot, Incubation

Journal: Cancers

Article Title: Define Critical Parameters of Trastuzumab-Mediated ADCC Assays via Assay Optimization Processes, Focusing on the Impact of Cryopreserved Effector Cells on Assay Performance.

doi: 10.3390/cancers16132367

Figure Lengend Snippet: Figure 3. Influence of the recovery culturing time and method on the extracellular release of gran- ulation proteins and cytokines secretion. (a–d) Comparison of the secretion levels of perforin (a), granzyme B (b), TNFα (c), and IFNγ (d) using ELISA in the 24 h cell culture media of the target cell SKBR-3 (T), the effector cells NK (E), coculture of SKBR-3 and NK (E+T), and trastuzumab-treated coculture of SKBR-3 and NK (Tras) with E/T ratio 5:1. Data in this figure were analyzed with Graph- Pad Prism using one-way ANOVA analysis with Tukey’s post hoc test (**** p < 0.0001). * Fresh NK cells were prepared from a different donor; thus, they were not included in statistical analysis.

Article Snippet: A FlowX Human NK Cell Phenotyping Flow Cytometry Kit (R&D Systems, Cat# FMC033, Minneapolis, MN, USA) was performed to assess NK cell phenotype using flow cytometry.

Techniques: Comparison, Enzyme-linked Immunosorbent Assay, Cell Culture

Journal: Cancers

Article Title: Define Critical Parameters of Trastuzumab-Mediated ADCC Assays via Assay Optimization Processes, Focusing on the Impact of Cryopreserved Effector Cells on Assay Performance.

doi: 10.3390/cancers16132367

Figure Lengend Snippet: Figure 4. The recovery culturing downregulates expression of CD16 and CD56 on NK cells. (a) Represen- tative immunoblot of CD16A (FcγRIIIa) in the whole cell lysates of NK cells isolated from unrecovered

Article Snippet: A FlowX Human NK Cell Phenotyping Flow Cytometry Kit (R&D Systems, Cat# FMC033, Minneapolis, MN, USA) was performed to assess NK cell phenotype using flow cytometry.

Techniques: Expressing, Western Blot, Isolation

Journal: Cancers

Article Title: Define Critical Parameters of Trastuzumab-Mediated ADCC Assays via Assay Optimization Processes, Focusing on the Impact of Cryopreserved Effector Cells on Assay Performance.

doi: 10.3390/cancers16132367

Figure Lengend Snippet: Figure 5. Phenotyping by flow cytometry indicates activation of NK cells by upregulation of CD69 during the recovery cultivation. (a) Flow cytometry analysis of surface CD69 expression in NK cells

Article Snippet: A FlowX Human NK Cell Phenotyping Flow Cytometry Kit (R&D Systems, Cat# FMC033, Minneapolis, MN, USA) was performed to assess NK cell phenotype using flow cytometry.

Techniques: Flow Cytometry, Activation Assay, Expressing

Journal: Cancers

Article Title: Define Critical Parameters of Trastuzumab-Mediated ADCC Assays via Assay Optimization Processes, Focusing on the Impact of Cryopreserved Effector Cells on Assay Performance.

doi: 10.3390/cancers16132367

Figure Lengend Snippet: Figure 6. Choice of target cell lines in the LDH ADCC assay and ADCC reporter assay. (a) Represen- tative immunoblots using lysates from HER2-positive cell lines; (b) Quantitation of the immunoblot; (c) Dose-dependent curves of trastuzumab-mediated LDH ADCC assay using SKBR-3, BT-474, and NCI-N87 cells. Target cells were treated with trastuzumab (0–1 µg/mL) and NK cells with E/T ratio 5:1 for 24 h and killing of SKBR-3 was measured with LDH ADCC assay; (c) Dose-dependent curves of trastuzumab-mediated ADCC activity using ADCC reporter assay in SKBR-3, BT-474 and NCI-N87 cells. Target cells were treated with trastuzumab (0–5 µg/mL) and Promega ADCC reporter cells with E/T ratio 6:1 and luminescence was measured after 6 h.

Article Snippet: A FlowX Human NK Cell Phenotyping Flow Cytometry Kit (R&D Systems, Cat# FMC033, Minneapolis, MN, USA) was performed to assess NK cell phenotype using flow cytometry.

Techniques: ADCC Assay, Reporter Assay, Western Blot, Quantitation Assay, Activity Assay

Journal: Cancers

Article Title: Define Critical Parameters of Trastuzumab-Mediated ADCC Assays via Assay Optimization Processes, Focusing on the Impact of Cryopreserved Effector Cells on Assay Performance.

doi: 10.3390/cancers16132367

Figure Lengend Snippet: Figure 7. Influence of assay buffer, target cell plating method, and treatment time on the measurement of ADCC of trastuzumab. (a) Luminescence fold changes from the LDH ADCC assay using RPMI-1640 with 1% BSA or 1% FBS. Data were analyzed with GraphPad Prism using two-way ANOVA with Šídák’s multiple comparisons test. p value < 0.0001 for 1% BSA vs. 1% FBS; (b) Luminescence fold changes using the LDH ADCC assay performed with either overnight or same-day culture of SKBR-3 target cells plated with 1% BSA or overnight culture of SKBR-3 in 10% FBS. Data were analyzed with GraphPad Prism using two-way ANOVA with Tukey’s multiple comparisons test. **** p < 0.0001. SKBR-3 cells were treated with trastuzumab (0–1 µg/mL) and NK cells with E/T ratio 5:1, and the killing of SKBR-3 was measured with the LDH ADCC assay, where “E” stands for effector-cell-only control.

Article Snippet: A FlowX Human NK Cell Phenotyping Flow Cytometry Kit (R&D Systems, Cat# FMC033, Minneapolis, MN, USA) was performed to assess NK cell phenotype using flow cytometry.

Techniques: ADCC Assay, Control

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Regnase-1 downregulation promotes pancreatic cancer through myeloid-derived suppressor cell-mediated evasion of anticancer immunity.

doi: 10.1186/s13046-023-02831-w

Figure Lengend Snippet: Fig. 6 Suppression of CTLs by CD11b+ MDSCs is responsible for the acceleration of tumor progression by Regnase-1 downregulation. A-D Evaluation of phenotypes of orthotopic syngeneic tumors of WT or Regnase-1 KO murine pancreatic cancer cells. Representative macro images of pancreatic tumors (A). Relative mRNA levels of Cd8a, Ifng, Fasl, and Gzmb (B) (N = 6 per group). Representative images of HE (C, left panel) and CD8a immunostaining (C, right panel) and the number of CD8-positive cells (C, right) (N = 6 per group). Dot plots of CD3+CD8+ cells evaluated by flow cytometry (D, left) and the proportion of CD8 + cells among CD45+ cells (D, right) (N = 3 per group). E–H Evaluation of phenotypes of orthotopic syngeneic tumors of WT or Regnase-1-KO murine pancreatic cancer cells with or without depletion of CD8+ cells upon anti-CD8a antibody or IgG treatment. Experimental schematic (E). Dot plots of CD3+ and CD8.+ cells in WT or Regnase-1-KO syngeneic tumors upon anti-CD8a antibody or IgG treatment evaluated by flow cytometry (F). Tumor weights (G) (N = 6 per group). The relative mRNA levels of Cd8a, Ifng, Fasl, and Gzmb (H) (N = 6 per group). Student’s t test was used to evaluate differences between 2 groups. One-way ANOVA with Tukey’s post hoc test was used to compare differences among 4 groups. *P < 0.05, scale bars: 100 μm (insets)

Article Snippet: BE0061, a fully neutralizing mAb recognizing CD8a, and control IgG were obtained from Bioxcell.

Techniques: Immunostaining, Flow Cytometry