biotin  (Miltenyi Biotec)

Journal: Molecular Therapy. Nucleic Acids

Article Title: Engineered mRNA backbones for gene expression in human T cells

doi: 10.1016/j.omtn.2026.102913

Figure Lengend Snippet: T cell-specific UTRs for optimized CAR expression, reactivity, and tonic signaling (A). Representative flow cytometry analysis of PBMC-derived T cells electroporated with CD19-CAR-encoding constructs using various 5′ UTRs. (B) Quantification of percentages of CD19-CAR + cells in (A). Data are normalized to the HBA1 UTR condition and presented as mean ± SEM from four independent experiments ( n = 4). Kruskal-Wallis test revealed no statistically significant difference ( p = 0.29). (C) MFI of CD19-CAR + cells in (A) normalized to the HBA1 UTR. Data represent four independent experiments ( n = 4). Kruskal-Wallis test indicated a significant difference among groups ( p < 0.005); Dunn’s post hoc test revealed significant reduction in the TNF-UTR group compared to HBA1 ( p = 0.0022). (D) Secreted IFN-γ levels in co-culture supernatants of PBMC-derived T cells electroporated with CD19-CAR mRNA using different 5′ UTRs and CD19 + NALM6 target cells. Data from two healthy donors (D29 and D40) are shown at various effector-to-target (E:T) ratios. (E) Left: interferon gamma ELISA in media taken from co-cultures of PBMC-derived T cells electroporated with CD19-CAR mRNA using different UTRs, either together with CD19 + (filled) or CD19 − (NALM6 KO, empty) at an E:T ratio of 4:1. Right: delta of interferon gamma secretion of E between the co-culture of electroporated T cells with CD19 + vs. CD19 − NALM6 cells. Bars represent mean ± SEM, n = 5 (CD19 + co-cultures) and n = 2 (CD19 − co-cultures) per construct. (F) Flow cytometry analysis of virus-specific T cells (VSTs) electroporated with mRNA constructs encoding for CD19-CAR using various UTRs. Note that cells are grown without target cells to demonstrate tonic signaling. (G) Pie charts of PD-1/TIM-3 population distribution of (F). Data are representative of two independent experiments.

Article Snippet: Cells were harvested 24–48 h post-electroporation, washed in FACS buffer (PBS with 2% FBS), and stained with the following fluorochrome-conjugated antibodies: CD19 CAR Detection Reagent, Biotin (Miltenyi Biotec, #130-129-550), followed by secondary staining with Anti-Biotin-APC (Miltenyi Biotec, REAfinity #130-113-854); TIM-3 APC-Cy7 (BioLegend, #345025); 4-1BB PE-Cy7 (BioLegend, #309820); Viability Dye eFluor 506/AmCyan (Thermo Fisher Scientific, #65-0866-14).

Techniques: Expressing, Flow Cytometry, Derivative Assay, Construct, Co-Culture Assay, Enzyme-linked Immunosorbent Assay, Virus

Journal: Frontiers in Oncology

Article Title: Clinical implementation of a one-step no-wash flow cytometry method allows for real-time monitoring of patients treated with autologous CAR-T cells

doi: 10.3389/fonc.2026.1774431

Figure Lengend Snippet: Determination of the limit of detection (LOD) and lower limit of quantification (LLOQ) for the two-step method. CAR-T cell absolute counts were measured in 31 negative control samples from patients not treated with CD19 CAR-T cells. The mean background signal is represented by the blue line. LOD was defined as mean + 3 standard deviations (SD), and LLOQ as mean + 10 SD.

Article Snippet: 100 μL of blood sample were stained with 5μL of CD19 CAR-T detection reagent (130-129–550 Miltenyi Biotec, Bergisch Gladbach, Germany) during 10 minutes at Room Temperature (RT) in the dark.

Techniques: Negative Control

Journal: Frontiers in Oncology

Article Title: Clinical implementation of a one-step no-wash flow cytometry method allows for real-time monitoring of patients treated with autologous CAR-T cells

doi: 10.3389/fonc.2026.1774431

Figure Lengend Snippet: Gating strategy for the single-step method. Absolute counting beads were excluded based on scatter and fluorescence properties. Dead cells were excluded using 7-aminoactinomycin D (7-AAD). CD45-positive leukocytes were selected, and lymphocytes were identified according to side scatter (SSC) characteristics. CD3-positive T cells were gated, and CAR-T cells were defined as viable CD45+/CD3+/CAR+ events using directly fluorochrome-conjugated CAR detection reagents (CD19 or BCMA). CD4 and CD8 subpopulations were subsequently identified within the CAR-positive T-cell compartment. Absolute quantification was calculated using TruCount beads according to the manufacturer’s formula.

Article Snippet: 100 μL of blood sample were stained with 5μL of CD19 CAR-T detection reagent (130-129–550 Miltenyi Biotec, Bergisch Gladbach, Germany) during 10 minutes at Room Temperature (RT) in the dark.

Techniques: Fluorescence, Quantitative Proteomics

Journal: Frontiers in Oncology

Article Title: Clinical implementation of a one-step no-wash flow cytometry method allows for real-time monitoring of patients treated with autologous CAR-T cells

doi: 10.3389/fonc.2026.1774431

Figure Lengend Snippet: Determination of LOD and LLOQ for the single-step method. CAR-T cell absolute counts were measured in 10 negative control samples from patients not treated with CD19 or BCMA CAR-T cells. The blue line represents the mean background signal. LOD was defined as mean + 3 SD and LLOQ (green dashed line) as mean + 10 SD.

Article Snippet: 100 μL of blood sample were stained with 5μL of CD19 CAR-T detection reagent (130-129–550 Miltenyi Biotec, Bergisch Gladbach, Germany) during 10 minutes at Room Temperature (RT) in the dark.

Techniques: Negative Control

Journal: bioRxiv

Article Title: Bilayer acoustic force spectroscopy (BAFS) for quantifying receptor-antigen binding strength in immune synapses

doi: 10.64898/2026.03.23.713630

Figure Lengend Snippet: a, BAFS sample preparation: step 1 – supported lipid bilayers are formed by injection of synthetic liposomes into AFS microfluidic chip; step 2 – passivated supported lipid bilayers are functionalized by injection of target ligand into the AFS chip; step 3 – after washing out excess ligand, sample cells (effector or control) are injected into the AFS chip; step 4 – sample cells are incubated on functionalized bilayer before washing out unadhered cells; step 5 – generation of acoustic standing waves, with a force ramp ranging from 0 to 1000 pN, leads to partial detachment of sample cells from the functionalized lipid bilayer. b, Schematic of cell attachment to functionalized SLBs due to formation of reconstituted immune synapses specifically through receptor-ligand interaction. c, Comparison of sample (CAR+ Jurkat T cells) and control (CAR- Jurkat T cells) at 0 pN (top) and 1000 pN (middle). CAR- Jurkat T cells are almost completely detached at 1000 pN, whereas most CAR+ Jurkat T cells are still attached at 1000 pN. Merge: green – detached cells collecting in acoustic nodes in the chip, magenta – cells attached at 0 pN but not 1000 pN, white – cells attached at 0 pN that remain attached at 1000 pN. d, Result of BAFS avidity measurement: average percentage of CAR+ Jurkat T cells (blue, n = 6 runs) and CAR- Jurkat T cells (red, n = 4 runs) bound to CD19+ SLB as a function of force. Shaded area: standard deviation. e, Schematic of Jurkat T cells expressing ZAP-70-eGFP in contact with SLBs functionalized with fluorescently labelled CD19. f, Example confocal microscopy images showing a CAR+ Jurkat T cell expressing ZAP-70-eGFP bound to CD19+ SLB after force application. ZAP-70-eGFP clusters in the effector cell (green, left) and SLB-bound CD19 clusters (magenta, middle) spatially colocalize (merge, right), indicating that increased binding is due to CD19-CAR interaction. g, Schematic of topographically complex cell-cell contact sites comprising not only specific receptor-ligand interactions, but also interactions between adhesion molecules and other off-target interactions. h, Result of cell-cell avidity measurement (classic AFS): average percentage of CAR+ Jurkat T cells (blue, n = 3 runs) and CAR- Jurkat T cells (red, n = 3 runs) bound to CD19-expressing Nalm-6 cells as a function of force. Shaded area: standard deviation. i, Boxplot comparing CAR+ Jurkat T-cell binding (blue, left) and CAR- Jurkat T-cell binding (red, left) to Nalm-6 cells at 1000 pN to CAR+ Jurkat T-cell binding (blue, right) and CAR- Jurkat T-cell binding (red, right) to CD19+ SLB at 1000 pN. j, Standard deviation of average binding at 1000 pN is decreased about 3-fold for CAR+ Jurkat T cells (blue) in BAFS as compared to AFS, and ∼29-fold for CAR- Jurkat T cells (red), due to abolished non-specific binding on the CD19+ SLB. k, Normalized pointwise signal-to-noise ratio is increased ∼10-fold in BAFS (SNR = 34.31) as compared to AFS (3.71). Dotted line: SNR=3 threshold (approximately 99.7% confidence that a detected signal is real and not a random fluctuation of background noise).

Article Snippet: CAT CAR+ Jurkat T cells were then sorted for high CAR expression as follows: CAT CAR-transduced Jurkat cells were prepared for sorting first by an incubation with a CD19 detection kit (CD19 CAR Detection Reagent, human, Biotin; catalog number 130-129-550, Miltenyi Biotec) according to manufacturer’s instructions.

Techniques: Sample Prep, Injection, Liposomes, Control, Incubation, Cell Attachment Assay, Comparison, Standard Deviation, Expressing, Confocal Microscopy, Binding Assay

Journal: bioRxiv

Article Title: Bilayer acoustic force spectroscopy (BAFS) for quantifying receptor-antigen binding strength in immune synapses

doi: 10.64898/2026.03.23.713630

Figure Lengend Snippet: a, Schematic demonstrating the sample preparation of parallel avidity measurements on several chips functionalized using the same protocol (top left) as compared to sequential avidity measurements on the same SLB-functionalized chip (bottom left). The corresponding average percentage of CAR+ Jurkat T cells bound to a 1% nickelated CD19+ SLB as a function of force (mean±SD) is displayed on the top right (dark blue) and bottom right (light blue), respectively. b, Boxplots displaying spread of percentage of cells bound at 1000 pN for sequential (left, light blue) and parallel avidity measurements (right, dark blue), corresponding to runs summarized in the average binding curves in , right. Box: 2 nd and 3 rd quartile, whiskers: 1 st and 4 th quartile. Red line: median. Data points in overlaid scatterplot are colored by run order, from dark to light gray. c, Top: Absolute difference of average percentage of cells bound as a function of force between sequential and parallel avidity runs. Bottom: coefficient of variation for parallel (dark blue) and sequential (light blue) BAFS avidity measurements, corresponding to the data displayed in the average binding curves in , right.

Article Snippet: CAT CAR+ Jurkat T cells were then sorted for high CAR expression as follows: CAT CAR-transduced Jurkat cells were prepared for sorting first by an incubation with a CD19 detection kit (CD19 CAR Detection Reagent, human, Biotin; catalog number 130-129-550, Miltenyi Biotec) according to manufacturer’s instructions.

Techniques: Sample Prep, Binding Assay

Journal: bioRxiv

Article Title: Bilayer acoustic force spectroscopy (BAFS) for quantifying receptor-antigen binding strength in immune synapses

doi: 10.64898/2026.03.23.713630

Figure Lengend Snippet: Fluorescence recovery after photobleaching (FRAP) of fluorescently labeled CD19 bound to a fluorescently labeled, partially nickelated SLB confirms bilayer and ligand mobility. a, Schematic illustrating the expected lateral diffusivity of CD19 ECD on the SLB, and the composition of the partially nickelated and partially fluorescently labelled SLB. b, Schematic illustrating a bleached spot in the SLB, as observed by total internal reflection fluorescence (TIRF) microscopy, and trajectories depicting diffusion of unbleached CD19 ECD into the bleached area. c, Quantification of FRAP of fluorescently labeled phospholipids in the SLB (cyan) and the His-tagged fluorescently labeled CD19 functionalized on the SLB (magenta) as a function of time. Intensity curves were double-normalized to an unbleached area of the SLB outside of the FRAP area.

Article Snippet: CAT CAR+ Jurkat T cells were then sorted for high CAR expression as follows: CAT CAR-transduced Jurkat cells were prepared for sorting first by an incubation with a CD19 detection kit (CD19 CAR Detection Reagent, human, Biotin; catalog number 130-129-550, Miltenyi Biotec) according to manufacturer’s instructions.

Techniques: Fluorescence, Labeling, Microscopy, Diffusion-based Assay

Journal: bioRxiv

Article Title: Bilayer acoustic force spectroscopy (BAFS) for quantifying receptor-antigen binding strength in immune synapses

doi: 10.64898/2026.03.23.713630

Figure Lengend Snippet: a, Schematic illustrating two-step functionalization of 1% biotinylated lipid-containing SLB with CD19 ECD. The lipid bilayer is first functionalized with neutravidin, which binds to the biotinylated phospholipid headgroups on the bilayer surface. In a second step, biotinylated CD19 ECD is flushed into the microfluidic AFS chip. Further sample preparation and avidity measurements are identical to those described in (see also Methods section). b, CAR+ and CAR- cell binding measured on 1% biotinylated SLBs displaying CD19 ECD. Average binding (solid lines) and standard deviation (shaded areas): CAR+ (black, n = 3 runs, sequential measurements) 61.0±4.0%, CAR- (red, n = 2 runs, sequential measurements) 0.8±0.6%.

Article Snippet: CAT CAR+ Jurkat T cells were then sorted for high CAR expression as follows: CAT CAR-transduced Jurkat cells were prepared for sorting first by an incubation with a CD19 detection kit (CD19 CAR Detection Reagent, human, Biotin; catalog number 130-129-550, Miltenyi Biotec) according to manufacturer’s instructions.

Techniques: Sample Prep, Binding Assay, Standard Deviation

Journal: bioRxiv

Article Title: Bilayer acoustic force spectroscopy (BAFS) for quantifying receptor-antigen binding strength in immune synapses

doi: 10.64898/2026.03.23.713630

Figure Lengend Snippet:

Article Snippet: CAT CAR+ Jurkat T cells were then sorted for high CAR expression as follows: CAT CAR-transduced Jurkat cells were prepared for sorting first by an incubation with a CD19 detection kit (CD19 CAR Detection Reagent, human, Biotin; catalog number 130-129-550, Miltenyi Biotec) according to manufacturer’s instructions.

Techniques: Comparison, Binding Assay

Journal: bioRxiv

Article Title: Bilayer acoustic force spectroscopy (BAFS) for quantifying receptor-antigen binding strength in immune synapses

doi: 10.64898/2026.03.23.713630

Figure Lengend Snippet: a, Unmasked nickelated lipids induce weak, non-specific binding of CAR+ Jurkat T cells. Right: Average percentage of CAR+ Jurkat T cells bound to CD19- SLB (gray, n = 4 runs) or SNAP-tag+ SLB (yellow, n = 2 runs) as a function of force. Shaded area: standard deviation. Inset: violin plots showing rupture force distribution for all cells tracked across the z-Movi BAFS measurements (gray: 1609 cells tracked in 4 separate runs, yellow: 1227 cells tracked in 2 separate runs); square – median unbinding force; dot – 90% unbinding force. b, Titration allows insights into ligand concentration-dependent binding effects. Average binding curves (mean±SD) of CAR+ Jurkat cells as a function of force on SLBs with 1% (dark blue, n = 6 runs), 0.1% (blue, n = 4 runs) and 0.01% (cyan, n = 3 runs) nickelated lipids, functionalized using the same concentration of His-tagged CD19 ECD. c, Average binding curves (mean±SD) of 1G4 αβTCR+ Jurkat T-cells on target Thp-1 cells after pulsing with cognate NY-ESO-1 antigen (black, n = 4 runs) and untreated Thp-1 cells (magenta, n = 4 runs). Deriving conclusion based on statistical analysis is rendered difficult due to the weak separation of the curves and large SD ( P =0.062, two-sample t-test, at 1000 pN). For detailed statistical analysis, see , Average binding curves (mean±SD) of 1G4 αβTCR+ Jurkat T-cells on NY-ESO-1 SCT+ SLBs (black, n = 3 runs) and αβTCR-Jurkat T-cells on NY-ESO-1 SCT+ SLBs (red, n = 4 runs). The separation at 1000 pN and small variation between runs for each condition permits clear separation of sample and control. e, Mean pointwise signal-to-noise ratio is increased ∼48-fold in BAFS (SNR = 43.12) as compared to AFS (0.89). Dotted line: SNR=3 threshold.

Article Snippet: CAT CAR+ Jurkat T cells were then sorted for high CAR expression as follows: CAT CAR-transduced Jurkat cells were prepared for sorting first by an incubation with a CD19 detection kit (CD19 CAR Detection Reagent, human, Biotin; catalog number 130-129-550, Miltenyi Biotec) according to manufacturer’s instructions.

Techniques: Binding Assay, Standard Deviation, Titration, Concentration Assay, Control