human simple plex assays  (Protein Simple Inc)

Journal: Journal of Translational Medicine

Article Title: Multi-omic profiling and preclinical efficacy of fratricide-driven, unedited CD7 CAR-T cells in T-cell leukemia

doi: 10.1186/s12967-026-07701-5

Figure Lengend Snippet: Tech transfer of UMCG-001 manufacturing to GMP-facility. ( A ) Overview for three UMCG-001 batches produced in a CliniMACS Prodigy ® bioreactor. Batch 1 and 3 were manufactured for 6 days, with cell harvest on day 6. Culture of batch 2 was expanded to day 12. For all, cell selection and activation were performed on day 0, LV transduction on day 1, HPL supplementation on day 4 and quality and characterization tests on harvesting day. Parallel lab batches were generated with matched-donor material for batch 2 and 3. ( B ) Flow cytometry-based quantification of transduction efficiency as %GFP + cells. ( C ) UMCG-001 expansion in million cells over time. Dotted line: DP release specification. ( D ) Fold-change expansion exhibited by UMCG-001 batch 3 generated at the GMP facility or Lab. ( E ) Fold-change expansion exhibited by UMCG-001 batch 2 generated at the GMP facility or Lab. ( F ) Flow cytometry scatterplots illustrating cell viability on harvesting day assessed by LIVE/DEAD TM staining within T-cell gate. ( G ) Histogram representation of CD7 expression on manufacturing day 6 among the different batches and CD19 CAR-T cells as reference. ( H ) Flow cytometry scatterplots representing CD7 vs. K12 CAR (GFP + ) expression across the batches on day 6. ( I ) Killing capacity across UMCG-001 batches of CD7 + and CD7 Neg cell lines at different E: T ratios for 24 h as AnnexinV/PI flow cytometry-based quantification. ( J ) Specific OV-CAR-3.Luc.CD7 killing calculated as indicated in Formula 1 by GMP batch 1 up to 5 rounds of 24 h-tumor re-challenges at different E: T ratios or ( K ) Percentage of viable cells at selected 2:1, 1:1 and 1:4 E: T. ( L ) IFN-γ secretion by the GMP batches when co-cultured with non-loaded (NL), CD7-loaded (CD7L), CD19-loaded (CD19L) or no beads for 24 h on day 6. Delta IFN-γ secretion in respect to NL beads. ( M ) IFN-γ secretion by UMCG-001 batches vs. CD19 CAR-T cells batches or ( N ) matched GMP and lab batch 3 as in ( L )

Article Snippet: IFN-γ secretion was quantified using the Simple Plex Human IFN-γ (3rd Gen) Cartridge on the ELLA platform (Protein Simple, Bio-Techne, Minneapolis, USA) as previously described [ ].

Techniques: Produced, Selection, Activation Assay, Transduction, Generated, Flow Cytometry, Staining, Expressing, Cell Culture

Journal: Journal of Translational Medicine

Article Title: Multi-omic profiling and preclinical efficacy of fratricide-driven, unedited CD7 CAR-T cells in T-cell leukemia

doi: 10.1186/s12967-026-07701-5

Figure Lengend Snippet: Autologous manufacturing and malignant contamination assessment in UMCG-001. ( A ) UMCG-001 manufactured from T-ALL patient 1 (T-ALL_1) derived material. ( B ) IFN-γ secretion upon 72 h co-culture of UMCG-001 batches with CD7 + (Jurkat, MOLT-4), autologous T-ALL and CD7 Neg K-562 cells. ( C ) Flow cytometry-based assessment of the percentage of killing of CD7 + (Jurkat, MOLT-4), autologous T-ALL and CD7 Neg K-562cells by UMCG-001. ( D ) Flow cytometry dot blot illustrating CD7 and surface CD3 (sCD3) expression in UMCG-001 and CD19 CAR-T cells upon fratricide. ( E ) Percentage of surface CD7 and CD3 over time in T.ALL_1 UMCG-001 final DP. Day − 1 represents the day before activation, day 0 after activation and transduction, and day 16 end of the fratricidal phase. ( F ) Schematic representation of sample preparation for subsequent single-cell RNA sequencing. T-ALL_1 original sample was composed of healthy (sCD3 + ) and blasts (sCD3 - ), which were sorted based on sCD3 expression. The whole sample was subjected to CAR - T manufacturing, after which CAR + cells were sorted based on GFP expression. All sorted samples were processed for V( D )J and gene expression (GEX) single-cell libraries. ( G ) Violin plot representation of genes related to malignant phenotype across TALL_1 sample. ( H ) Relative abundance of TCR clones across CD19 CAR-T cells UMCG-001 and sCD3 + samples, calculated as the number of unique TCRs per total number of cells. ( I ) Clone sizes across CD19 CAR-T cells and UMCG-001 batches generated from independent healthy donors (gray) and T-ALL_1 (black). The number of cells per clone group is shown

Article Snippet: IFN-γ secretion was quantified using the Simple Plex Human IFN-γ (3rd Gen) Cartridge on the ELLA platform (Protein Simple, Bio-Techne, Minneapolis, USA) as previously described [ ].

Techniques: Derivative Assay, Co-Culture Assay, Flow Cytometry, Dot Blot, Expressing, Activation Assay, Transduction, Sample Prep, Single Cell, RNA Sequencing, Gene Expression, Clone Assay, Generated

Journal: bioRxiv

Article Title: Convergent Roles of Growth Differentiation Factor-15 (GDF-15) in Mechanotransduction, Vascular Disorganization, and Immune Suppression in Melanoma

doi: 10.64898/2026.01.14.699463

Figure Lengend Snippet: (A) Mechanical force exerted by applied weight on cells was used to examine pressure effects during metastatic processes. ECM stiffness was studied by comparing a rigid ( G ’ ≈ 100 kPa) environment resembling the acral melanoma model with a softer environment (G’ ≈ 10 kPa) in a GelMA hydrogel mimicking cutaneous melanoma. The color gradient (yellow to black) represents GDF-15 expression levels, with higher expression in the acral melanoma model. (B) Dynamic moduli (storage modulus, G ′, and loss modulus, G ″) of soft and stiff bulk hydrogel scaffolds, made with 4 and 15% w/v GelMA concentrations, respectively, versus the angular frequency at a constant oscillatory strain (∼ 0.1%). (C) G ′ and (D) G ″ of soft and stiff scaffolds at a constant angular frequency (1 rad s -1 ) and constant oscillation strain (0.1%) (n = 5). (E) GelMA droplets (4 or 15% w/v) in oil and surfactant are stored at 4 °C to create physically crosslinked microgels, followed by photo-crosslinking to fabricate thermally stable GelMA microgels. (F) Brightfield microscopy images of soft and stiff microgels incubated in 37°C DPBS for 60 min, imaged at 15-min intervals. Scale bar is 100 µm. (G) Size distribution of soft and stiff microgels after incubation at 37 °C for 60 min ( n > 800). A one-way analysis of variance (ANOVA), followed by Tukey’s post hoc multiple comparisons test, was performed to compare the study groups.

Article Snippet: 24 h later, conditioned media were collected from the plates and analyzed by GDF-15 ELISA for quantification of protein levels in cell culture media and serum samples, using the DOST ELISA for human GDF-15 (R&D Systems) according to the manufacturer’s protocol.

Techniques: Expressing, Microscopy, Incubation

Journal: bioRxiv

Article Title: Convergent Roles of Growth Differentiation Factor-15 (GDF-15) in Mechanotransduction, Vascular Disorganization, and Immune Suppression in Melanoma

doi: 10.64898/2026.01.14.699463

Figure Lengend Snippet: Representative immunofluorescence images showing GDF-15 expression (yellow), nuclei (green), and F-actin (magenta) in melanoma cells ( A : UACC 903; B : A375P; C : WM4235) cultured with soft ( G ’ ≈ 10 kPa) and stiff ( G ’ ≈ 100 kPa) microgel matrices. Increased GDF-15 expression is observed in the stiff microenvironment, resembling conditions found in acral melanoma. (D) Quantification of GDF-15 fluorescence intensity in melanoma cells (Cutaneous: UACC 903 & A375P; Acral: WM4235) cultured in soft and stiff matrices. Box graphs represent median ± SEM. (E) Western blot analysis of GDF-15 expression in melanoma cells (UACC 903 & A375P) under soft or stiff GelMA ECM matrix conditions. ERK2 serves as a loading control, and GDF-15 protein levels are quantified and normalized to ERK2. Cells in the stiff matrix show increased GDF-15 expression compared to the soft matrix. (F, G, H) Modeling GelMA microgel stiffness in melanoma tumors. (F, G) Representative immunofluorescence images showing GDF-15 (yellow), nuclei (blue), and F-actin (magenta) in tumor sections (F: UACC 903; G: A375P) after 7 days of growth in mice. (H) Quantification of GDF-15 fluorescence intensity, equivalent to expression in tumor cells after growing on stiff or soft microgels.

Article Snippet: 24 h later, conditioned media were collected from the plates and analyzed by GDF-15 ELISA for quantification of protein levels in cell culture media and serum samples, using the DOST ELISA for human GDF-15 (R&D Systems) according to the manufacturer’s protocol.

Techniques: Immunofluorescence, Expressing, Cell Culture, Fluorescence, Western Blot, Control

Journal: bioRxiv

Article Title: Convergent Roles of Growth Differentiation Factor-15 (GDF-15) in Mechanotransduction, Vascular Disorganization, and Immune Suppression in Melanoma

doi: 10.64898/2026.01.14.699463

Figure Lengend Snippet: (A) GDF-15 expression in UACC 903 and B16F10 cells grown on collagen ECM or in suspension compared to adherent control conditions. Western blot analysis shows that both altered ECM context and anchorage independence induce GDF-15 expression. α-Tubulin was used as a loading control. ( B-1) Schematic of the “sandwich” setup in which cells are first grown on glass coverslips, then the coverslip is inverted onto a glass–plastic support to position the cell layer between rigid surfaces. (B-2) Schematic of the weight-based compression assay in which defined masses are placed on the upper glass layer to apply graded normal forces and generate increasing compressive pressures on the cell monolayer. (C) Western blot analysis of GDF-15 expression in UACC 903 cells exposed to increasing mechanical pressure (22 Pa, 38 Pa, 54 Pa, 100 Pa), with the flipped coverslip alone (22 Pa) serving as a low-pressure condition and UACC 903M (red) as a positive control. (D) Quantification of fold change in GDF-15 expression relative to the uncompressed negative control in UACC 903 cells, demonstrating a pressure-dependent increase in GDF-15. (E) Western blot analysis of GDF-15 expression in B16F10 cells under the same graded mechanical pressures, with UACC 903M (red) used as a positive control. (F) Quantification of fold change in GDF-15 expression relative to the uncompressed negative control in B16F10 cells, showing a similar pressure-dependent induction of GDF-15.

Article Snippet: 24 h later, conditioned media were collected from the plates and analyzed by GDF-15 ELISA for quantification of protein levels in cell culture media and serum samples, using the DOST ELISA for human GDF-15 (R&D Systems) according to the manufacturer’s protocol.

Techniques: Expressing, Suspension, Control, Western Blot, Positive Control, Negative Control

Journal: bioRxiv

Article Title: Convergent Roles of Growth Differentiation Factor-15 (GDF-15) in Mechanotransduction, Vascular Disorganization, and Immune Suppression in Melanoma

doi: 10.64898/2026.01.14.699463

Figure Lengend Snippet: (A) GDF-15 combined with either bradykinin (BK) or VEGF markedly increases intercellular gaps in confluent HUVEC monolayers compared to control, with red arrows indicating gap edges and asterisks marking representative gaps (40× magnification). (B) Quantification of intercellular gap numbers per field of view in HUVEC monolayers treated with increasing concentrations of BK (B1) or VEGF (B2) in the presence or absence of GDF-15, demonstrating a synergistic effect of GDF-15 with each permeability mediator on gap formation. (C) Schematic illustrating how GDF-15, together with BK or VEGF, enhances myosin light chain kinase (MLCK)-driven phosphorylation of myosin light chain (pMLC), thereby increasing actomyosin contractility and destabilizing VE-cadherin-based adherens junctions to promote gap formation. (D) Western blot analysis of HUVEC lysates showing that combined treatment with GDF-15 and BK or VEGF elevates pMLC levels relative to single treatments, consistent with enhanced contractility and increased endothelial gap formation. In-vivo permeability changes by GDF-15 and permeability mediators

Article Snippet: 24 h later, conditioned media were collected from the plates and analyzed by GDF-15 ELISA for quantification of protein levels in cell culture media and serum samples, using the DOST ELISA for human GDF-15 (R&D Systems) according to the manufacturer’s protocol.

Techniques: Control, Permeability, Phospho-proteomics, Western Blot, In Vivo

Journal: bioRxiv

Article Title: Convergent Roles of Growth Differentiation Factor-15 (GDF-15) in Mechanotransduction, Vascular Disorganization, and Immune Suppression in Melanoma

doi: 10.64898/2026.01.14.699463

Figure Lengend Snippet: (A) Representative Miles assay images showing Evans Blue dye accumulation in murine ears treated with GDF-15, PAF, or their combination, with GDF-15 plus PAF producing the most significant leakage, which is abrogated by co-treatment with the S1PR1 agonist ASR396. (B) Quantification of Evans Blue extracted from ear tissue, confirming that GDF-15 potentiates PAF-induced vascular leakage and that ASR396 reverses this effect. (C) Representative single-micro-vessel experiment in which rat mesenteric venules were sequentially perfused with control BSA Ringer’s solution, GDF-15 (10 µg/mL), and then GDF-15 plus PAF (10 nM), illustrating dynamic changes in hydraulic conductivity ( L p ) over time. (D) Summary of micro-vessel permeability measurements showing that GDF-15 alone does not significantly increase L p , but markedly augments the PAF-induced rise in L p , from baseline to approximately 50-fold over control ( L p test / L p CTRL ). (E) Confocal VE-cadherin staining of fixed mesenteric micro-vessels revealing continuous junctions in control vessels, junctional disruptions after PAF, and extensive junctional breaks and gaps after combined GDF-15 plus PAF, consistent with the magnitude of permeability increase (scale bar, 40 µm). (F,G) ELISA of conditioned media demonstrating reduced GDF-15 secretion in A375M shGDF-15 and UACC 903M shGDF-15 cells compared with their respective parental lines. (H,I) Quantification of tumor cell retention in the lungs 24 hours after intravenous injection, showing that PAF enhances lung retention of parental A375M and UACC 903M cells but not GDF-15 knockdown cells, indicating that GDF-15 is required for PAF-driven metastatic extravasation.

Article Snippet: 24 h later, conditioned media were collected from the plates and analyzed by GDF-15 ELISA for quantification of protein levels in cell culture media and serum samples, using the DOST ELISA for human GDF-15 (R&D Systems) according to the manufacturer’s protocol.

Techniques: Control, Permeability, Staining, Enzyme-linked Immunosorbent Assay, Injection, Knockdown

Journal: bioRxiv

Article Title: Convergent Roles of Growth Differentiation Factor-15 (GDF-15) in Mechanotransduction, Vascular Disorganization, and Immune Suppression in Melanoma

doi: 10.64898/2026.01.14.699463

Figure Lengend Snippet: (A) A375M and (B) UACC 903M parental cells expressing GDF-15 formed 60-70% larger tumors compared to shGDF-15 counterparts. Three representatives of (C) A375 M and (D) UACC 903M parental cells, compared with shGDF-15 tumors, showed gross differences in size and bloodiness. (E) Tumors shown in (C and D) were processed for H&E stain, and the areas of tumor hemorrhagic vascular morphology were quantified, showing significantly less in the parental tumors compared to the GDF-15 knockdown tumors. (F) Levels of GDF-15 in serum measured by ELISA increased with the size of A375M and UACC 903M tumors. (G) GDF-15 decreased NK cell-mediated killing of tumor cells. Target UACC 903M parental or shGDF-15 cells were labeled with 5 µM Cell Trace Violet (CTV) and then combined with NK-92 cells at the indicated effector to target ratios (E:T) in triplicate for 5 h. Cultures were stained with 7AAD for 10 min at room temperature, and the percentage of live target cells (7AAD-negative, CTV+) in each culture was quantified by flow cytometry. UACC 903M parental and shGDF-15 tumors from nude mice were subjected to NK cell profiling. (H) Significantly higher percentages of NK cells were present in the shGDF-15 tumors. (I) Higher NK cell activity was also observed in the shGDF-15 tumors. CD11b was used as an NK activation marker.

Article Snippet: 24 h later, conditioned media were collected from the plates and analyzed by GDF-15 ELISA for quantification of protein levels in cell culture media and serum samples, using the DOST ELISA for human GDF-15 (R&D Systems) according to the manufacturer’s protocol.

Techniques: Expressing, Staining, Knockdown, Enzyme-linked Immunosorbent Assay, Labeling, Flow Cytometry, Activity Assay, Activation Assay, Marker