plate bound anti cd3  (Miltenyi Biotec)

Journal: Science Advances

Article Title: BLIMP1 negatively regulates IL-2 signaling in T cells

doi: 10.1126/sciadv.adx8105

Figure Lengend Snippet: Purified CD4 + T cells from frozen PBMCs of HDs and patients with acute ATL were ex vivo stimulated with anti-CD3 and anti-CD28 and IL-2 for 24 hours at 37°C and stained for flow cytometry. Cells were gated as CD3 + CD4 + CD25 + CCR4 + T cells (ATL-like T reg cells). ( A ) Schematic representation of the experiment ( B to K ). Created in BioRender. S. Roy (2025) https://BioRender.com/ae8e1h0 . Representative histograms with cumulative statistics for flow cytometry analysis of [(B) and (C)] CD25 and [(D) and (E)] CD122 expression. [(F) and (G)] Representative histograms and cumulative statistics for FOXP3 expression by flow cytometry. [(H) and (I)] Flow cytometric analysis of pSTAT5, pAKT, and pERK showing representative histograms and cumulative statistics. [(J) and (K)] Representative histograms and statistics for BLIMP1 expression by flow cytometry. Data are representative of means ± SEM from three independent experiments ( n = 4 to 5 individuals). Two-tailed unpaired Student’s t test was used for statistical analysis. ( L ) Schematic representation of PRDM1 overexpression in ATL cells. Created in BioRender. S. Roy (2025) https://BioRender.com/ae8e1h0 . ( M ) Histograms showing protein expression for BLIMP1, CD25, CD122, pSTAT5, pAKT, and pERK in ATL cells transduced with pLVX-EV (empty vector control) or pLVX- PRDM1 (vector expressing PRDM1 ). ( N ) Statistical analysis showing mean fluorescent intensity (MFI) for BLIMP1, CD25, CD122, pSTAT5, pAKT, and pERK, protein levels in ATL cells transduced with pLVX-EV (empty vector control) or pLVX- PRDM1 (vector expressing PRDM1 ). Data are representative of means ± SEM from two independent experiments ( n = 5 individuals). Two-tailed paired Student’s t test was used for statistical analysis.

Article Snippet: CD4 + T cells were then activated with plate-bound anti-human CD3 (2 μg/ml, #BE0001-2; clone OKT3, BioXCell) and soluble anti-human CD28 (1 μg/ml; #BE0248; clone 9.3, BioXCell) and cultured in RPMI 1640 medium with 10% FBS, 2 mM l -glutamine, and penicillin (50 U/ml) + streptomycin (50 μg/ml) in the presence of recombinant human IL-2 (100 IU/ml) for 72 hours at 37°C.

Techniques: Purification, Ex Vivo, Staining, Flow Cytometry, Expressing, Two Tailed Test, Over Expression, Transduction, Plasmid Preparation, Control

Journal: Science Advances

Article Title: BLIMP1 negatively regulates IL-2 signaling in T cells

doi: 10.1126/sciadv.adx8105

Figure Lengend Snippet: Purified CD4 + T cells from HDs ( n = 3) and patients with acute ATL ( n = 5) were stimulated with anti-CD3 and anti-CD28 in presence of IL-2 for 4 hours at 37°C. The cells were harvested and subjected to scRNA-seq. ( A ) Schematic representation of the experimental setup for ( B to H ). Created in BioRender. S. Roy (2025) https://BioRender.com/9zdi679 . (B) Uniform Manifold Approximation and Projection (UMAP) plot showing all cells split by HD cells (left) and ATL cells (right). (C) Bar plot showing proportion of the cells in each cluster annotated by Azimuth for each HD and ATL sample. (D) UMAP plot showing distribution of T reg cells split by HD cells (left) and ATL cells (right). (E) Violin plot showing PRDM1 expression in T reg cells in HDs and patients with ATL. (F) UMAP plot showing distribution of PRDM1 − versus PRDM1 + T reg populations in HDs and patients with ATL. (G) Pie chart showing percentage of cells in HDs and patients with ATL that express any amount of PRDM1 . (H) Bar plot showing top pathways (by z score) from Qiagen IPA using differentially expressed genes derived from PRDM1 − versus PRDM1 + populations within T reg cells cluster in patients with ATL.

Article Snippet: CD4 + T cells were then activated with plate-bound anti-human CD3 (2 μg/ml, #BE0001-2; clone OKT3, BioXCell) and soluble anti-human CD28 (1 μg/ml; #BE0248; clone 9.3, BioXCell) and cultured in RPMI 1640 medium with 10% FBS, 2 mM l -glutamine, and penicillin (50 U/ml) + streptomycin (50 μg/ml) in the presence of recombinant human IL-2 (100 IU/ml) for 72 hours at 37°C.

Techniques: Purification, Expressing, Derivative Assay

Journal: Science Advances

Article Title: BLIMP1 negatively regulates IL-2 signaling in T cells

doi: 10.1126/sciadv.adx8105

Figure Lengend Snippet: Purified CD4 + T cells from frozen PBMCs of HDs and patients with acute ATL were ex vivo stimulated with anti-CD3 and anti-CD28 and IL-2 for 24 hours at 37°C and stained for flow cytometry. Cells were gated as CD3 + CD4 + CD25 + CCR4 + T cells (ATL-like T reg cells). ( A ) Schematic representation of the experiment ( B to K ). Created in BioRender. S. Roy (2025) https://BioRender.com/ae8e1h0 . Representative histograms with cumulative statistics for flow cytometry analysis of [(B) and (C)] CD25 and [(D) and (E)] CD122 expression. [(F) and (G)] Representative histograms and cumulative statistics for FOXP3 expression by flow cytometry. [(H) and (I)] Flow cytometric analysis of pSTAT5, pAKT, and pERK showing representative histograms and cumulative statistics. [(J) and (K)] Representative histograms and statistics for BLIMP1 expression by flow cytometry. Data are representative of means ± SEM from three independent experiments ( n = 4 to 5 individuals). Two-tailed unpaired Student’s t test was used for statistical analysis. ( L ) Schematic representation of PRDM1 overexpression in ATL cells. Created in BioRender. S. Roy (2025) https://BioRender.com/ae8e1h0 . ( M ) Histograms showing protein expression for BLIMP1, CD25, CD122, pSTAT5, pAKT, and pERK in ATL cells transduced with pLVX-EV (empty vector control) or pLVX- PRDM1 (vector expressing PRDM1 ). ( N ) Statistical analysis showing mean fluorescent intensity (MFI) for BLIMP1, CD25, CD122, pSTAT5, pAKT, and pERK, protein levels in ATL cells transduced with pLVX-EV (empty vector control) or pLVX- PRDM1 (vector expressing PRDM1 ). Data are representative of means ± SEM from two independent experiments ( n = 5 individuals). Two-tailed paired Student’s t test was used for statistical analysis.

Article Snippet: Purified CD4 + T cells were activated with plate-bound anti-mouse CD3 (2 μg/ml, #BE0001-1; clone 145-2C11, BioXCell) and soluble anti-mouse CD28 (1 μg/ml, #BE0015-1; clone 37.51, BioXCell) and cultured in RPMI 1640 medium (#11875093, Gibco) supplemented with 10% fetal bovine serum (FBS) (#100-106, GeminiBio), 2 mM l -glutamine (#25030-149, Gibco), penicillin (50 U/ml) + streptomycin (50 μg/ml) (#15070-063, Gibco), and 50 μM 2-mercaptoethanol (#21985-023, Gibco) for 72 hours in the presence of recombinant human IL-2 (100 IU/ml; #Ro 23-6019, Roche) at 37°C after which the cells were harvested and assayed.

Techniques: Purification, Ex Vivo, Staining, Flow Cytometry, Expressing, Two Tailed Test, Over Expression, Transduction, Plasmid Preparation, Control

Journal: Science Advances

Article Title: BLIMP1 negatively regulates IL-2 signaling in T cells

doi: 10.1126/sciadv.adx8105

Figure Lengend Snippet: Purified CD4 + T cells from HDs ( n = 3) and patients with acute ATL ( n = 5) were stimulated with anti-CD3 and anti-CD28 in presence of IL-2 for 4 hours at 37°C. The cells were harvested and subjected to scRNA-seq. ( A ) Schematic representation of the experimental setup for ( B to H ). Created in BioRender. S. Roy (2025) https://BioRender.com/9zdi679 . (B) Uniform Manifold Approximation and Projection (UMAP) plot showing all cells split by HD cells (left) and ATL cells (right). (C) Bar plot showing proportion of the cells in each cluster annotated by Azimuth for each HD and ATL sample. (D) UMAP plot showing distribution of T reg cells split by HD cells (left) and ATL cells (right). (E) Violin plot showing PRDM1 expression in T reg cells in HDs and patients with ATL. (F) UMAP plot showing distribution of PRDM1 − versus PRDM1 + T reg populations in HDs and patients with ATL. (G) Pie chart showing percentage of cells in HDs and patients with ATL that express any amount of PRDM1 . (H) Bar plot showing top pathways (by z score) from Qiagen IPA using differentially expressed genes derived from PRDM1 − versus PRDM1 + populations within T reg cells cluster in patients with ATL.

Article Snippet: Purified CD4 + T cells were activated with plate-bound anti-mouse CD3 (2 μg/ml, #BE0001-1; clone 145-2C11, BioXCell) and soluble anti-mouse CD28 (1 μg/ml, #BE0015-1; clone 37.51, BioXCell) and cultured in RPMI 1640 medium (#11875093, Gibco) supplemented with 10% fetal bovine serum (FBS) (#100-106, GeminiBio), 2 mM l -glutamine (#25030-149, Gibco), penicillin (50 U/ml) + streptomycin (50 μg/ml) (#15070-063, Gibco), and 50 μM 2-mercaptoethanol (#21985-023, Gibco) for 72 hours in the presence of recombinant human IL-2 (100 IU/ml; #Ro 23-6019, Roche) at 37°C after which the cells were harvested and assayed.

Techniques: Purification, Expressing, Derivative Assay