Journal: STAR Protocols

Article Title: Protocol for 3D-guided sectioning and deep cell phenotyping via light sheet imaging and 2D spatial multiplexing

doi: 10.1016/j.xpro.2025.104296

Figure Lengend Snippet: 3D light sheet and 2D multi-cyclic imaging data comparison (Human OvCa) (A) Imaris 3D surface rendering of autofluorescence (cyan) and CD326 positive cells (red). (B) Imaris 3D surface rendering of autofluorescence (cyan) with target plane in yellow. (C) Light sheet guided target plane selection representing CD326 positive cell (purple), CD45 positive cells (red), and CD3 positive cells (green). (D) DAPI overview image of selected tissue slice for 2D MACSima™ imaging. (E) Magnified merged six color multiparameter MICS image with CD45 (green), CD326 (cyan), FOLR1 (purple), Collagen III (red), Collagen IV (red), and CD31 (yellow). (F–L) Single staining MICS images (white) of DAPI (F), CD45 (G), CD326 (H), FOLR1 (I), Collagen III (J), Collagen IV (K), and CD31 (L) (gray) (see “Antibodies”). Scale bars: (A–F) 1 mm; (E) 250 μm; (F–L) 500 μm.

Article Snippet: CD3 antibody, anti-human, Vio G570, REAfinity , Miltenyi Biotec B.V. & Co. KG , Cat# 130-133-790 RRID: AB_3664347.

Techniques: Imaging, Comparison, Selection, Staining

Journal: Journal of Nanobiotechnology

Article Title: MiR-1290 in natural killer cell derived extracellular vesicles: a pathogenic mediator of lupus nephritis and therapeutic target for th17 regulation

doi: 10.1186/s12951-026-04212-9

Figure Lengend Snippet: Characterization of NK-EVs and their effect on Th17 cell differentiation in vitro. (a) Schematic of the experimental procedure for treating primary T cells with NK-EVs. (b) Purity analysis of sorted CD4 + T cells by flow cytometry. (c) Purity analysis of sorted NK cells, showing the proportion of CD3 − CD56 + NK cells exceeded 94%. (d) Size distribution of NK-EVs as determined by NTA. (e) Western blot analysis of exosomal markers and specific cargos in NK-EVs, with 293T-derived EVs as a control. (f) TEM analysis of NK-EVs showing cup-shaped bilayer membrane structures (indicated by black arrows, scale bar: 200 nm). (g) Immunofluorescence staining showing the uptake of NK-EVs by T cells (indicated by black arrows, scale bar: 10 μm) and (h) fluorescence intensity analysis of the local region indicated by white arrows. (i) Gating strategy and representative flow cytometry plots for CD4 + IL-17 A + T cells under Th17-polarizing conditions after EVs treatment. (j) LN-EVs significantly reduced the proportion of CD4 + IL-17 A + cells ( N = 3). (k) Schematic of the procedures for generating Protein-free NK-EVs via protease treatment and RNA-free NK-EVs via RNase treatment. (l) Effects of LN-derived NK-EVs, Protein-free NK-EVs, and RNA-free NK-EVs on the proportion of Th17 cell differentiation. (m) Effects of LN-derived NK-EVs, Protein-free NK-EVs, and RNA-free NK-EVs on the IL17A mRNA level in T cells. (n) Effects of LN-derived NK-EVs, Protein-free NK-EVs, and RNA-free NK-EVs on the levels of inflammatory cytokines IFN-γ, TNF-α, IL-17 A, and IL-10 in T cell culture supernatants. Data are presented as mean ± SD ( N = 3). * P < 0.05, ** P < 0.01 (One-way ANOVA)

Article Snippet: Subsequently, CD3+ T cells and CD3-depleted lymphocytes were obtained by MACS with mouse CD3 microbeads (Miltenyi Biotec, #130-094-973).

Techniques: Cell Differentiation, In Vitro, Flow Cytometry, Western Blot, Derivative Assay, Control, Membrane, Immunofluorescence, Staining, Fluorescence, Cell Culture

Journal: Journal of Nanobiotechnology

Article Title: MiR-1290 in natural killer cell derived extracellular vesicles: a pathogenic mediator of lupus nephritis and therapeutic target for th17 regulation

doi: 10.1186/s12951-026-04212-9

Figure Lengend Snippet: Characterization and distribution of engineered NK-EVs. (a) NTA size distribution analysis of NK-EVs before and after engineering. (b) TEM analysis of NK-EVs before and after engineering. Scale bar: 100 nm. (c) Zeta potential measurement of NK-EVs before and after engineering ( N = 3). (d) Uptake of engineered EVs by mouse splenic lymphocytes, CD3 + T cells, and CD3-depleted lymphocytes (scale bar: 10 μm). (e) Representative in vivo images showing the temporal biodistribution of EVs. Mice received an intravenous injection of DiD-labeled EVs. Fluorescence images were acquired at the indicated time points (2, 4, 8, and 24 h) post-injection. (f) Representative ex vivo images of dissected major organs at corresponding time points. The color scale represents radiant efficiency ([p/s/cm²/sr]/[µW/cm²]). Data are presented as mean ± SD. * P < 0.05

Article Snippet: Subsequently, CD3+ T cells and CD3-depleted lymphocytes were obtained by MACS with mouse CD3 microbeads (Miltenyi Biotec, #130-094-973).

Techniques: Zeta Potential Analyzer, In Vivo, Injection, Labeling, Fluorescence, Ex Vivo

Journal: Cell Transplantation

Article Title: A novel GMP-manufactured medicinal product candidate composed of NK and γδ T cells as adjunct immunotherapy for hematopoietic stem cell transplantation

doi: 10.1177/09636897251374248

Figure Lengend Snippet: Flow cytometric analysis of cell populations during the process. Panel A. Number of cells and proportion of populations obtained at the different steps of the process starting from buffy coat (BC) or leukapheresis (LA). Panels B–D. One representative experiment using LA (#3) as staring material is shown. Panel B. CD14 + monocytes (a), CD19 + B cells (b) CD3 + T cells (c), CD3 − CD56 + NK cells (d), γδ and αβ T lymphocytes (e) as well as Vδ1 and Vδ2 subsets (f), present after Ficoll-Paque gradient in gated lympho-mononuclear cells. Panels C and D. CD14 + monocytes (a), CD19 + B cells (b) CD3 − CD56 + NK cells (c), γδ and αβ T lymphocytes (d) as well as Vδ1 and Vδ2 subsets (e), gated mononuclear cells at the end of expansion (day 14) (Panel C) and harvested after αβ T-cell depletion (Panel D). Panel E shows γδ T cell differentiation, by APC-CD27 and PEVio770-CD45RA staining.

Article Snippet: Immunophenotype was assessed on at least 50,000 cells by flow cytometry using fluorochrome-conjugated monoclonal antibodies (mAb): FITC-CD3, PerCP-CD14, Viogreen-CD19, PEVio770 anti-TCRαβ, APC anti-TCRγδ, Viogreen-TCRVδ1, Vioblue-TCRVδ2, and APC-CD56 (all from Miltenyi).

Techniques: Cell Differentiation, Staining

Journal: Cell Transplantation

Article Title: A novel GMP-manufactured medicinal product candidate composed of NK and γδ T cells as adjunct immunotherapy for hematopoietic stem cell transplantation

doi: 10.1177/09636897251374248

Figure Lengend Snippet: Expression of CD16, natural cytotoxic receptors and exhaustion markers. Panel a. Expression of CD16, NKG2A, NKG2D, NKp30, NKp44 and NKp46 was tested by flow cytometry and analyzed in gated lympho-mononuclear cells. Experiments performed using batch from LA#4 is reported. NK and γδ T cells were discriminated by the different expression of CD3 (negative in NK and positive in γδ T lymphocytes). Results from all batches produced are reported in Panel b. Expression of PD-1 and TIM-3 in γδ (gated in CD3 + cells) and NK cells (gated in CD3 − ) present in batch #4 is shown in Panel c. Percentages of the two exhaustion markers expression in all batches produced are detailed in Panel d.

Article Snippet: Immunophenotype was assessed on at least 50,000 cells by flow cytometry using fluorochrome-conjugated monoclonal antibodies (mAb): FITC-CD3, PerCP-CD14, Viogreen-CD19, PEVio770 anti-TCRαβ, APC anti-TCRγδ, Viogreen-TCRVδ1, Vioblue-TCRVδ2, and APC-CD56 (all from Miltenyi).

Techniques: Expressing, Flow Cytometry, Produced

Journal: Cell Transplantation

Article Title: A novel GMP-manufactured medicinal product candidate composed of NK and γδ T cells as adjunct immunotherapy for hematopoietic stem cell transplantation

doi: 10.1177/09636897251374248

Figure Lengend Snippet: Cytotoxic activity of the GADEKILL against target tumor cells. CD107a surface expression on γδ T lymphocytes (Panel a), Vδ2 cells (Panel b), and NK cells (Panel c) in the absence (ctr) or presence of target cells (K562, SK-N-AS and SK-N-SH) have been analyzed on the four batches. γδ T cells and Vδ2 cytotoxic subsets were analyzed in gated CD45 + CD3 + cells, by staining with APC-TCRγδ, Vioblue anti-Vδ2. NK were analyzed on gated CD45 + CD3 − cells, by staining with APC-CD56 mAb. CD107a degranulation assay was performed using a 1:1 E:T ratio. Horizontal bars indicated medians. Asterisks indicated statistically significant differences. One representative experiment from batch #2 is shown in panels d, e and f. Degranulation of effector cells in the absence of the targets (Panel d) and against the myeloid leukemia K562 cells (Panel e) and the neuroblastoma SK-N-AS cell line (Panel f) is reported.

Article Snippet: Immunophenotype was assessed on at least 50,000 cells by flow cytometry using fluorochrome-conjugated monoclonal antibodies (mAb): FITC-CD3, PerCP-CD14, Viogreen-CD19, PEVio770 anti-TCRαβ, APC anti-TCRγδ, Viogreen-TCRVδ1, Vioblue-TCRVδ2, and APC-CD56 (all from Miltenyi).

Techniques: Activity Assay, Expressing, Staining, Degranulation Assay