Journal: Microsystems & Nanoengineering

Article Title: High-fidelity bioassembly of organoids and spheroids using inertial droplet microfluidics for precision oncology and tumor microenvironment modeling

doi: 10.1038/s41378-026-01244-x

Figure Lengend Snippet: The automated OsciSphere bioassembly platform and workflow. a 3D model of the OsciSphere platform. The inset shows the core 8-channel droplet generator, which integrates an oscillator, syringe pump, disposable pipette tips, and a cooling block to maintain Matrigel in a liquid state. b Photograph of the automated workstation layout. c Schematic of the OsciSphere workflow. 1) Cells are suspended in Matrigel at 4 °C. 2) The platform’s droplet generator dispenses uniform droplets into a 96-well plate containing a biphasic carrier oil/culture medium overlay. 3) Droplets are solidified at 37 °C for 10 min. 4) Solidified microspheres are transferred into the underlying medium using an antistatic gun or gentle agitation. 5) This automated process yields arrays of µMCTs or µTDOs ready for high-throughput screening applications. d Time-lapse imaging (left) demonstrates the “pull-break-sediment” cycle of droplet formation. The platform reliably produces approximately 100 uniform 30 nL droplets from only 3 µL of Matrigel (right), achieving excellent size uniformity. Scale bar, 500 µm. e Encapsulation uniformity demonstrated by fluorescent beads within Matrigel microspheres (60.9 ± 5.1 beads per droplet, CV = 7.67%, n = 28). f Platform versatility is shown by forming uniform microspheres from agarose and HAMA. g Radar chart comparing OsciSphere’s performance metrics (e.g., uniformity, throughput, automation) against conventional 3D culture methods. OsciSphere enables rapid and uniform 3D culture, including ( h ) µMCTs and i µTDOs. j Dome TDOs exhibit significant spatial heterogeneity, leading to diffusion-limited regions and apoptotic cores by Day 3

Article Snippet: Growth and morphology were monitored via time-lapse phase-contrast imaging (Incucyte S3, Sartorius, Göttingen, Germany).

Techniques: Transferring, Blocking Assay, Gentle, High Throughput Screening Assay, Imaging, Encapsulation, Diffusion-based Assay

Journal: Microsystems & Nanoengineering

Article Title: High-fidelity bioassembly of organoids and spheroids using inertial droplet microfluidics for precision oncology and tumor microenvironment modeling

doi: 10.1038/s41378-026-01244-x

Figure Lengend Snippet: OsciSphere operates in a deterministic regime to produce uniform µMCTs that recapitulate key physiological tumor features. a Scaffold-free ULA culture (Day 4) yields a multimodal population, with a single large central spheroid and numerous highly variable satellite aggregates. b Conventional Matrigel dome culture (Day 3) exhibits significant spatial heterogeneity driven by diffusion gradients, with smaller spheroids in the nutrient-poor center and larger ones at the periphery. c OsciSphere-generated µMCTs (~600 cells/droplet, Day 3) exhibit structural isotropy and consistency. d Quantification of spheroid diameters confirms the superior monodispersity of µMCTs ( n = 100, CV = 4.2%) compared to the high variability of ULA ( n = 244) and dome ( n = 237) cultures (mean ± SD, **** p < 0.0001). e Bright-field time-lapse shows rapid self-assembly of compact HCT116 spheroids by Day 1. f Live/Dead staining at 72 h confirms high cell viability. g Histological analysis (H&E) reveals a dense 3D tissue architecture, with immunohistochemistry for Ki67 confirming robust proliferative activity within the spheroid. h Optimization of formation efficiency reveals a critical density threshold at 600–900 cells per microsphere, achieving 99.6% successful formation by Day 2 ( n = 10, mea n ± SD, red star indicates the optimal 600-cell condition). i Immunofluorescence (IF) of 2D HCT116 cultures shows basal expression of Vimentin and N-cadherin. Scale bar, 10 µm. j µMCTs display pronounced, organized expression of mesenchymal markers (Vimentin, N-cadherin), indicative of EMT. Scale bar, 50 µm. k This invasive phenotype is validated by RT-qPCR, showing significant upregulation of key EMT-associated genes ( N-cadherin , Snail , Slug ) and cancer stem cell-associated genes ( Sox2 , Oct4 ) in µMCTs ( n = 3, mea n ± SD, **** p < 0.0001). l µMCTs exhibit a physiologically relevant reduced proliferation rate compared to 2D cultures, mimicking in vivo tumor kinetics ( n = 5, mean ± SEM, **** p < 0.0001). m Flow cytometry analysis reveals elevated intracellular ROS levels in µMCTs (51.2%) versus 2D cultures (30.2%), consistent with the establishment of metabolic gradients and a hypoxic tumor microenvironment. Statistical significance was analyzed by using one-way analysis of variance (ANOVA)

Article Snippet: Growth and morphology were monitored via time-lapse phase-contrast imaging (Incucyte S3, Sartorius, Göttingen, Germany).

Techniques: Diffusion-based Assay, Generated, Staining, Immunohistochemistry, Activity Assay, Immunofluorescence, Expressing, Quantitative RT-PCR, In Vivo, Flow Cytometry

Journal: Microsystems & Nanoengineering

Article Title: High-fidelity bioassembly of organoids and spheroids using inertial droplet microfluidics for precision oncology and tumor microenvironment modeling

doi: 10.1038/s41378-026-01244-x

Figure Lengend Snippet: OsciSphere-derived µTDOs resolve diffusion limitations to drive superior growth and maturation. a Comparative time-lapse microscopy reveals the impact of culture geometry. Conventional Matrigel domes exhibit severe spatial heterogeneity: while organoids at the nutrient-rich “Edge” grow, those in the diffusion-limited “Core” undergo apoptosis (red arrow) by Day 3. In contrast, OsciSphere µTDOs (bottom row) exhibit uniform, necrosis-free growth independent of spatial position. b Quantification of projected surface area demonstrates significantly accelerated expansion kinetics for µTDOs compared to dome cultures ( n = 58, mean ± SEM, **** p < 0.0001). c Morphogenic analysis reveals enhanced maturation in the OsciSphere format, with a significantly higher frequency of multi-budded organoids observed by Day 2 ( n = 58, mea n ± SEM). d Viability imaging (Calcein-AM/PI) on Day 3 confirms that µTDOs maintain high cell survival without the central necrosis observed in static hydrogel cultures. e Histological validation against native murine intestine. H&E staining demonstrates that µTDOs recapitulate the polarized crypt-villus architecture of the in vivo epithelium. Immunohistochemistry for Ki67 (brown) confirms the preservation of active proliferative zones in the crypt domains of both µTDOs and native tissue. Statistical significance was analyzed by using one-way ANOVA

Article Snippet: Growth and morphology were monitored via time-lapse phase-contrast imaging (Incucyte S3, Sartorius, Göttingen, Germany).

Techniques: Derivative Assay, Diffusion-based Assay, Time-lapse Microscopy, Imaging, Biomarker Discovery, Staining, In Vivo, Immunohistochemistry, Preserving

Journal: Microsystems & Nanoengineering

Article Title: High-fidelity bioassembly of organoids and spheroids using inertial droplet microfluidics for precision oncology and tumor microenvironment modeling

doi: 10.1038/s41378-026-01244-x

Figure Lengend Snippet: OsciSphere enables high-fidelity modeling of the patient-specific tumor-immune microenvironment . a Schematic of the precision immuno-oncology workflow. Patient-derived hCRC tissues are processed into µPDOs via OsciSphere, creating physically permissive scaffolds that support autologous PBMC infiltration. b Bright-field micrographs of source PDO lines established from three independent hCRC patients. c Transcriptomic validation confirms high-fidelity modeling: established PDOs (O) maintain strong gene expression correlations ( R > 0.87) with their matched parental tumor tissue (T). d Genomic profiling demonstrates that µPDOs preserve the patient-specific mutational landscape across a panel of key oncogenic drivers. e Histological comparison reveals that µPDOs (bottom) recapitulate the native tumor architecture (top). H&E staining shows comparable morphology, while Ki67 staining confirms the maintenance of robust proliferative zones in both the parent tissue (brown) and the µPDOs (red). f Failure mode of conventional culture: endpoint imaging reveals that the dense, large-volume Matrigel dome acts as a physical barrier, excluding PBMCs (red) from the tumor core. g OsciSphere overcomes the barrier effect: time-lapse imaging captures the active migration of PBMCs (red) into the µPDO (dashed circle), facilitating sustained tumor-immune interactions (yellow arrows) over 72 h. Scale bar, 100 µm. h Flow cytometry analysis quantifies IFN-γ levels. i Quantif i cation of the mean fluorescence intensity of CD8 + IFN-γ + PBMCs from PBMC-only, PBMCs + µPDOs, PBMC + µPDOs + sintilimab groups ( n = 3, mea n ± SD; * p < 0.05, ** p < 0.01, ns, not significant). Statistical significance was analyzed by using one-way ANOVA

Article Snippet: Growth and morphology were monitored via time-lapse phase-contrast imaging (Incucyte S3, Sartorius, Göttingen, Germany).

Techniques: Derivative Assay, Biomarker Discovery, Gene Expression, Comparison, Staining, Imaging, Migration, Flow Cytometry, Fluorescence

Journal: bioRxiv

Article Title: Multifaceted immune resistance landscapes in human oligodendrocytes protect against cytotoxic T cells and are dysregulated in MS brain cell subsets

doi: 10.64898/2026.04.21.719872

Figure Lengend Snippet: (A-B) Generation of HLA-A2 + Luciferase + human oligodendrocytic cell line MO3.13-A2-Luc. (A-B) FACS analysis of the (A) HLA-A2 expression on wildtype (WT) MO3.13 cells (left), MO3.13-A2 cells 2 weeks after transfection with HLA-A2 expression vector (middle), and MO3.13-A2 cells after the FACS sorting for HLA-A2 (right). (B) GFP expression on MO3.13-A2-Luc cells after lentiviral transduction (left), GFP expression on FACS sorted MO3.13-A2-Luc cells (middle) and HLA-A2 expression on MO3.13-A2-Luc cells (right). Grey histograms represent the isotype control and untransduced cells for the analysis of HLA-A2 and GFP expression respectively. (C-E) Optimization of siRNA reverse transfection protocol for MO3.13-A2-Luc cells. (C) Real-time live cell imaging of MO3.13-A2-Luc cells transfected either with a non-targeting siRNA control (Scr) or with a siRNA cocktail targeting genes essential for cell survival (cell death siRNA, siCD). Transfected cells were imaged via the IncuCyte SX5 system for 72 h and real-time % cell confluency was quantified. (D) RT-qPCR analysis of PDL1 mRNA expression in MO3.13-A2-Luc cells transfected either with Scr siRNA or pool of 4 non-overlapping siRNAs targeting PDL1 (siCD274). Results are presented as fold change compared to the Scr after β-actin mRNA normalization. (E) FACS analysis of PD-L1 surface expression on MO3.13-A2-Luc cells transfected either with Scr or PD-L1 specific siRNA pool. Left: representative histograms indicating % PD-L1 expression, right: mean fluorescent intensity (MFI) of PD-L1 from 2 independent FACS data. (F) Phenotypic characterization of FluTC. The expression of CD4 and CD8 was determined on CD3 + FluTC, whereas the expression of effector and memory markers CD45RO and CD62L and co-inhibitory immune checkpoint molecules PD-1, LAG-3 and TIM-3 was determined on CD3 + CD8 + FluTC. (Tn: naïve T cells, Tcm: central memory T cells, Teff: terminal effector T cells, Tem: effector memory T cells). Grey histograms represent the isotype control. (G) Luciferase-based cytotoxicity assay to optimize the co-culture conditions for MO3.13-A2-Luc and FluTC. MO3.13-A2-Luc were pulsed with serial dilutions of the flu-peptide and co-cultured with FluTC. After 20 h of co-culture, remaining relative luciferase units (RLU) were measured. RLU measured in pulsed samples were normalized to RLU of unpulsed (no peptide) control. Statistical significance was calculated compared with no peptide control. (H) Selection of cytotoxicity and viability controls for the HTP screen. MO3.13-A2-Luc cells were transfected with the indicated siRNAs. After 72 h of transfection cells were pulsed with 0,01 µg/ml flu-peptide and co-cultured either with FluTC or treated with control T cell medium (CM). 20 h following co-culture, remaining RLU was measured. The RLU of each sample was normalized to the Scr1 control in the cytotoxicity (FluTC) or viability (CM) setting to determine the impact of gene knockdown. (A-G) Representative data of at least 2 independent experiments. (H) Cumulative data of three independent experiments each performed with four replicates per sample. (D, G-H) Graphs show mean +/- SD. p-values were calculated using two-tailed student’s t-test. * = p < 0.05, ** = p < 0.01, *** = p < 0.005, **** = p < 0.001.

Article Snippet: Apoptosis of MO3.13-A2 was determined by real-time live-cell imaging using Incucyte® SX5 live cell imager (Sartorius).

Techniques: Luciferase, Expressing, Transfection, Plasmid Preparation, Transduction, Control, Live Cell Imaging, Quantitative RT-PCR, Cytotoxicity Assay, Co-Culture Assay, Cell Culture, Selection, Knockdown, Two Tailed Test

Journal: bioRxiv

Article Title: Multifaceted immune resistance landscapes in human oligodendrocytes protect against cytotoxic T cells and are dysregulated in MS brain cell subsets

doi: 10.64898/2026.04.21.719872

Figure Lengend Snippet: (A) Expression of selected HITs in MO3.13-A2-Luc cells. Conventional PCR was performed to detect gene expression of selected HITs in undifferentiated and differentiated MO3.13-A2-Luc cells. Myelin basic protein (MBP) expression was used as positive control for MO3.13 oligodendrocyte differentiation and β-actin was used as house-keeping gene. (B) RT-qPCR analysis of STK11, KCNH8, ABCA2, SLC1A3 and CHRNA1 mRNA expression in MO3.13-A2-Luc cells transfected either with Scr siRNA or pool of 30 siRNAs targeting corresponding genes. Results are presented as fold change compared to the Scr after β-actin mRNA normalization. (C) Luminex-based cytokine analysis of the FluTC – MO3.13-A2-Luc cell co-cultures. MO3.13-A2-Luc cells transfected either with Scr siRNA or IRG-specific pool of 30 siRNAs and co-cultured with FluTC for 24 h. TNFα, IFNγ, GM-CSF, CXCL10, IL-13, MIP-1α, MIP-1β, IL-6 and IL-8 levels were depicted. Each line represents an independent experiment; values indicate the average of triplicates. (D-E) Selected IRGs protect MO3.13 oligodendrocytes against cytotoxic molecules secreted by activated T cells. MO3.13-A2-Luc cells transfected either with Scr siRNA or IRG-specific siRNA pool and treated with the supernatant of CD3/CD28-activated FluTC. (D) MO3.13 oligodendrocyte survival was determined by luciferase-based cytotoxicity assay. (E) Real-time cytotoxicity assay (IncuCyte® SX5 System) to analyze activated supernatant-induced MO3.13 oligodendrocyte apoptosis over 24 h in MO3.13-A2 cells. Cells were transfected and treated as in (D). Incucyte® Cytotox-Red Dye was added as an indicator of apoptosis. The graph shows total red object integrated intensity per well (RCU x µm²/Image). (A, D-E) Representative data of at least three independent experiments. (B-C) Cumulative data of three and five independent experiments respectively. Values represent (B, D-E) the mean ± SD, (C) mean. P-value was calculated using paired two-tailed Student’s t-test (* = p < 0.05, ** = p < 0.01, *** = p < 0.005, **** = p < 0.001).

Article Snippet: Apoptosis of MO3.13-A2 was determined by real-time live-cell imaging using Incucyte® SX5 live cell imager (Sartorius).

Techniques: Expressing, Gene Expression, Positive Control, Quantitative RT-PCR, Transfection, Luminex, Cell Culture, Luciferase, Cytotoxicity Assay, Two Tailed Test

Journal: bioRxiv

Article Title: Multifaceted immune resistance landscapes in human oligodendrocytes protect against cytotoxic T cells and are dysregulated in MS brain cell subsets

doi: 10.64898/2026.04.21.719872

Figure Lengend Snippet: (A) FACS analysis to determine the surface expression of IFNG-R1, TNF-R1, TNF-R2, FAS, TRAIL-R1, and TRAIL-R2 in MO3.13-A2-Luc cells. Grey histograms represent the isotype control. (B) Impact of IRG knockdown on TRAIL-R2 surface expression in MO3.13-A2-Luc cells. Left panel: Representative overlay of histograms to compare TRAIL-R2 expression on IRG +/- MO3.13-A2-Luc cells. Right panel: Compiled data of mean fluorescence intensity (MFI) for TRAIL-R2 expression. (C) Real-time cytotoxicity assay (IncuCyte® SX5 System) to analyze TRAIL-induced apoptosis over 48 hours in IRG +/- MO3.13-A2-Luc cells. Incucyte® Cytotox-Red Dye was added to the transfected MO3.13 oligodendrocytes together with TRAIL treatment as an indicator of apoptosis. The graph shows total red object integrated intensity per well (RCU x µm²/Image). (D) Western blot analysis of total/cleaved caspase-3/8/9, TRAF2, phosho(p)/total TAK1, MKK4, JNK, NF-κB, pLKB1 and Bcl2 in IRG +/- MO3.13-A2-Luc cells upon 4 h treatment with TRAIL. Experiment was run in two separate blots each including Scr samples. Each HIT was shown in comparison to Scr sample analyzed in the same blot (Blot1: Scr, siSTK11, siKCNH8 and siABCA2; Blot2: Scr, siSLC1A3 and siCHRNA1). Representative data of (A & D) two, (B-C) three independent experiments. Values represent the mean ± SD. P-value was calculated using paired two-tailed Student’s t-test (* = p < 0.05, ** = p < 0.01, *** = p < 0.005, **** = p < 0.001).

Article Snippet: Apoptosis of MO3.13-A2 was determined by real-time live-cell imaging using Incucyte® SX5 live cell imager (Sartorius).

Techniques: Expressing, Control, Knockdown, Fluorescence, Cytotoxicity Assay, Transfection, Western Blot, Comparison, Two Tailed Test

Journal: bioRxiv

Article Title: Multifaceted immune resistance landscapes in human oligodendrocytes protect against cytotoxic T cells and are dysregulated in MS brain cell subsets

doi: 10.64898/2026.04.21.719872

Figure Lengend Snippet: (A-B) Generation of HLA-A2 + Luciferase + human oligodendrocytic cell line MO3.13-A2-Luc. (A-B) FACS analysis of the (A) HLA-A2 expression on wildtype (WT) MO3.13 cells (left), MO3.13-A2 cells 2 weeks after transfection with HLA-A2 expression vector (middle), and MO3.13-A2 cells after the FACS sorting for HLA-A2 (right). (B) GFP expression on MO3.13-A2-Luc cells after lentiviral transduction (left), GFP expression on FACS sorted MO3.13-A2-Luc cells (middle) and HLA-A2 expression on MO3.13-A2-Luc cells (right). Grey histograms represent the isotype control and untransduced cells for the analysis of HLA-A2 and GFP expression respectively. (C-E) Optimization of siRNA reverse transfection protocol for MO3.13-A2-Luc cells. (C) Real-time live cell imaging of MO3.13-A2-Luc cells transfected either with a non-targeting siRNA control (Scr) or with a siRNA cocktail targeting genes essential for cell survival (cell death siRNA, siCD). Transfected cells were imaged via the IncuCyte SX5 system for 72 h and real-time % cell confluency was quantified. (D) RT-qPCR analysis of PDL1 mRNA expression in MO3.13-A2-Luc cells transfected either with Scr siRNA or pool of 4 non-overlapping siRNAs targeting PDL1 (siCD274). Results are presented as fold change compared to the Scr after β-actin mRNA normalization. (E) FACS analysis of PD-L1 surface expression on MO3.13-A2-Luc cells transfected either with Scr or PD-L1 specific siRNA pool. Left: representative histograms indicating % PD-L1 expression, right: mean fluorescent intensity (MFI) of PD-L1 from 2 independent FACS data. (F) Phenotypic characterization of FluTC. The expression of CD4 and CD8 was determined on CD3 + FluTC, whereas the expression of effector and memory markers CD45RO and CD62L and co-inhibitory immune checkpoint molecules PD-1, LAG-3 and TIM-3 was determined on CD3 + CD8 + FluTC. (Tn: naïve T cells, Tcm: central memory T cells, Teff: terminal effector T cells, Tem: effector memory T cells). Grey histograms represent the isotype control. (G) Luciferase-based cytotoxicity assay to optimize the co-culture conditions for MO3.13-A2-Luc and FluTC. MO3.13-A2-Luc were pulsed with serial dilutions of the flu-peptide and co-cultured with FluTC. After 20 h of co-culture, remaining relative luciferase units (RLU) were measured. RLU measured in pulsed samples were normalized to RLU of unpulsed (no peptide) control. Statistical significance was calculated compared with no peptide control. (H) Selection of cytotoxicity and viability controls for the HTP screen. MO3.13-A2-Luc cells were transfected with the indicated siRNAs. After 72 h of transfection cells were pulsed with 0,01 µg/ml flu-peptide and co-cultured either with FluTC or treated with control T cell medium (CM). 20 h following co-culture, remaining RLU was measured. The RLU of each sample was normalized to the Scr1 control in the cytotoxicity (FluTC) or viability (CM) setting to determine the impact of gene knockdown. (A-G) Representative data of at least 2 independent experiments. (H) Cumulative data of three independent experiments each performed with four replicates per sample. (D, G-H) Graphs show mean +/- SD. p-values were calculated using two-tailed student’s t-test. * = p < 0.05, ** = p < 0.01, *** = p < 0.005, **** = p < 0.001.

Article Snippet: Apoptosis of MO3.13-A2 was determined by real-time live-cell imaging using Incucyte® SX5 live cell imager (Sartorius).

Techniques: Luciferase, Expressing, Transfection, Plasmid Preparation, Transduction, Control, Live Cell Imaging, Quantitative RT-PCR, Cytotoxicity Assay, Co-Culture Assay, Cell Culture, Selection, Knockdown, Two Tailed Test

Journal: bioRxiv

Article Title: Multifaceted immune resistance landscapes in human oligodendrocytes protect against cytotoxic T cells and are dysregulated in MS brain cell subsets

doi: 10.64898/2026.04.21.719872

Figure Lengend Snippet: (A) Expression of selected HITs in MO3.13-A2-Luc cells. Conventional PCR was performed to detect gene expression of selected HITs in undifferentiated and differentiated MO3.13-A2-Luc cells. Myelin basic protein (MBP) expression was used as positive control for MO3.13 oligodendrocyte differentiation and β-actin was used as house-keeping gene. (B) RT-qPCR analysis of STK11, KCNH8, ABCA2, SLC1A3 and CHRNA1 mRNA expression in MO3.13-A2-Luc cells transfected either with Scr siRNA or pool of 30 siRNAs targeting corresponding genes. Results are presented as fold change compared to the Scr after β-actin mRNA normalization. (C) Luminex-based cytokine analysis of the FluTC – MO3.13-A2-Luc cell co-cultures. MO3.13-A2-Luc cells transfected either with Scr siRNA or IRG-specific pool of 30 siRNAs and co-cultured with FluTC for 24 h. TNFα, IFNγ, GM-CSF, CXCL10, IL-13, MIP-1α, MIP-1β, IL-6 and IL-8 levels were depicted. Each line represents an independent experiment; values indicate the average of triplicates. (D-E) Selected IRGs protect MO3.13 oligodendrocytes against cytotoxic molecules secreted by activated T cells. MO3.13-A2-Luc cells transfected either with Scr siRNA or IRG-specific siRNA pool and treated with the supernatant of CD3/CD28-activated FluTC. (D) MO3.13 oligodendrocyte survival was determined by luciferase-based cytotoxicity assay. (E) Real-time cytotoxicity assay (IncuCyte® SX5 System) to analyze activated supernatant-induced MO3.13 oligodendrocyte apoptosis over 24 h in MO3.13-A2 cells. Cells were transfected and treated as in (D). Incucyte® Cytotox-Red Dye was added as an indicator of apoptosis. The graph shows total red object integrated intensity per well (RCU x µm²/Image). (A, D-E) Representative data of at least three independent experiments. (B-C) Cumulative data of three and five independent experiments respectively. Values represent (B, D-E) the mean ± SD, (C) mean. P-value was calculated using paired two-tailed Student’s t-test (* = p < 0.05, ** = p < 0.01, *** = p < 0.005, **** = p < 0.001).

Article Snippet: Apoptosis of MO3.13-A2 was determined by real-time live-cell imaging using Incucyte® SX5 live cell imager (Sartorius).

Techniques: Expressing, Gene Expression, Positive Control, Quantitative RT-PCR, Transfection, Luminex, Cell Culture, Luciferase, Cytotoxicity Assay, Two Tailed Test

Journal: bioRxiv

Article Title: Multifaceted immune resistance landscapes in human oligodendrocytes protect against cytotoxic T cells and are dysregulated in MS brain cell subsets

doi: 10.64898/2026.04.21.719872

Figure Lengend Snippet: (A) FACS analysis to determine the surface expression of IFNG-R1, TNF-R1, TNF-R2, FAS, TRAIL-R1, and TRAIL-R2 in MO3.13-A2-Luc cells. Grey histograms represent the isotype control. (B) Impact of IRG knockdown on TRAIL-R2 surface expression in MO3.13-A2-Luc cells. Left panel: Representative overlay of histograms to compare TRAIL-R2 expression on IRG +/- MO3.13-A2-Luc cells. Right panel: Compiled data of mean fluorescence intensity (MFI) for TRAIL-R2 expression. (C) Real-time cytotoxicity assay (IncuCyte® SX5 System) to analyze TRAIL-induced apoptosis over 48 hours in IRG +/- MO3.13-A2-Luc cells. Incucyte® Cytotox-Red Dye was added to the transfected MO3.13 oligodendrocytes together with TRAIL treatment as an indicator of apoptosis. The graph shows total red object integrated intensity per well (RCU x µm²/Image). (D) Western blot analysis of total/cleaved caspase-3/8/9, TRAF2, phosho(p)/total TAK1, MKK4, JNK, NF-κB, pLKB1 and Bcl2 in IRG +/- MO3.13-A2-Luc cells upon 4 h treatment with TRAIL. Experiment was run in two separate blots each including Scr samples. Each HIT was shown in comparison to Scr sample analyzed in the same blot (Blot1: Scr, siSTK11, siKCNH8 and siABCA2; Blot2: Scr, siSLC1A3 and siCHRNA1). Representative data of (A & D) two, (B-C) three independent experiments. Values represent the mean ± SD. P-value was calculated using paired two-tailed Student’s t-test (* = p < 0.05, ** = p < 0.01, *** = p < 0.005, **** = p < 0.001).

Article Snippet: Apoptosis of MO3.13-A2 was determined by real-time live-cell imaging using Incucyte® SX5 live cell imager (Sartorius).

Techniques: Expressing, Control, Knockdown, Fluorescence, Cytotoxicity Assay, Transfection, Western Blot, Comparison, Two Tailed Test

Journal: bioRxiv

Article Title: Multifaceted immune resistance landscapes in human oligodendrocytes protect against cytotoxic T cells and are dysregulated in MS brain cell subsets

doi: 10.64898/2026.04.21.719872

Figure Lengend Snippet: (A-B) Impact of SIK3 on TRAIL-mediated MO3.13-A2-Luc killing was determined by (A) Luciferase-based cytotoxicity assay as described in supp and (B) real-time cytotoxicity assay as described in . Representative data of (A) three (B) two independent experiments. Values represent the mean ± SD. P-value was calculated using paired two-tailed Student’s t-test (* = p < 0.05, ** = p < 0.01).

Article Snippet: Apoptosis of MO3.13-A2 was determined by real-time live-cell imaging using Incucyte® SX5 live cell imager (Sartorius).

Techniques: Luciferase, Cytotoxicity Assay, Two Tailed Test