anti-hu cd25 apc clone: bc96  (Thermo Fisher)

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Enhanced T cell activation and cytotoxicity against AML via targeted anti-CD99 nanoparticle treatment

doi: 10.1016/j.biopha.2024.117265

Figure Lengend Snippet: α-CD99-A192 increases expression of activation marker CD38 but does not affect T cell major phenotypes. Flow cytometry panels of control (untreated and A192), and α-CD99-A192 treated PBMCs for A, CD3 , B, CD4 , C, CD8 and D, CD25. Flow cytometry panels of control (untreated and A192), and α-CD99-A192 treated expanded T cells for E, CD3 , F, CD4 , G, CD8 and H, CD25. I, Quantified percentage levels of CD3, CD4, CD8 and CD25 untreated, A192 treated, and α-CD99-A192 treated PBMCs. J, Quantified percentage levels of CD3, CD4, CD8 and CD25 untreated, A192 treated, and α-CD99-A192 treated T cells. K, Flow cytometry panel of CD3 + CD69 + cells (Q2) for control A192 treated T cells and α-CD99-A192 treated T cells. L, Flow cytometry panel of CD3 + CD38 + cells (Q2) for control A192 treated T cells and α-CD99-A192 treated T cells. M, Percentage of CD69 + for A192 and α-CD99-A192 treated t cells where no significant change is observed in the α-CD99-A192 treated T cells compared with the control. N, Percentage of CD38 + for A192 and α-CD99-A192 treated T cells where a significant increase is observed in the α-CD99-A192 treated T cells compared with the control. The differences between the groups were analyzed using unpaired t tests (**,P<0.01; *,P<0.05). The experiments were performed utilizing samples obtained from three donors for T cell phenotyping and five donors for CD69 and CD38 activation marker analysis. The T cells utilized in these experiments were expanded using PHA and IL2 from the PBMCs obtained from healthy donors.

Article Snippet: The master mix was prepared using anti-human CD3 FITC (Clone: UCHT1; Biolegend, Cat#300406), Anti-Human CD4 PE (Clone: OKT4; eBioscience Cat#4337556), Anti-Hu CD8a PE-Cyanine 7 (Clone: SK1; eBioscience Cat#2066348) and Anti-Hu CD25 APC (Clone: BC96; eBioscience Cat#1946588) antibodies.

Techniques: Expressing, Activation Assay, Marker, Flow Cytometry, Control

Journal: iScience

Article Title: Single cell profiling of hematopoietic stem cell transplant recipients reveals TGF-β1 and IL-2 confer immunoregulatory functions to NK cells

doi: 10.1016/j.isci.2024.111416

Figure Lengend Snippet: CD56 bright NK cells expanded in IL-2 and TGF-β1 exhibit regulatory functions in vitro (A) Method of NK cell isolation, PBMCs were stained with FITC conjugated antibodies for the following lineage markers: CD3, CD4, CD8α, CD14, CD15, CD19, CD20, TCRαβ, TCRδγ, CD33, CD34, CD203c, FCεr1α, CD79α, and CD138. NK were sorted as live CD56 bright CD16 − or CD56 dim CD16 + , and CCR6 − and CRTH2 − to exclude ILC2s and ILC3s. (B) Representative gating strategy to sort CD56 bright CD16 − NK cells and CD56 dim CD16 + NK cells from healthy PBMCs. (C) Fold expansion of NK cells after 19 days of in vitro culture in IL-2, IL-15, and IL-18 cytokines, or IL-2, TGF-β1, and anti-IFN-γ referred to as NK conv and NK TGF-β , respectively. (D) Representative and summary cytokine expression of CD56 bright NK conv and NK TGF- β at day 20 post-expansion, stimulated with phorbol 12-mystrate 13-acetate (PMA)/ionomycin for a duration of 6 h ( n = 7, 3). (E) Active TGF-β1 within supernatants of NK cells plated at 1 M/mL at D20 ( n = 14, 16). (F and G) Naive autologous CD4 + T cells were labeled with cell proliferation dye (CPD) for a short term 4-day culture with either NK conv ( n = 4) or NK TGF-β ( n = 6) with anti-CD3 and anti-CD28 antibodies. Suppression was measured by percent divided of the live CD3 + CD4 + CD56 − CD8 − in comparison to naive CD4 + T cells activated with anti-CD3 and anti-CD28 antibodies. Dashed line indicates 0 percent suppression. (H) Summary of CPD + HELIX-NP + and Annexin-V + K562 cells. Helix-NP + Annexin-V + indicates apoptotic cells ( n = 5, 6). (I) Expression of FOXP3 and CD25 within co-cultures with NK cells ( n = 3–6). (J) Following 20 days co-culture of CD4 + T cells with IL-2 and TGF-β1-treated CD56 bright NK cells, CD25 + CD127 low/− T cells were sorted then co-cultured with CPD stained allogeneic naive T cells for 3 days ( n = 3, 4). Induced Tregs (iTregs) were generated using standard induction protocols of IL-2 and TGF-β1 ( n = 3, 4). Suppression as measured by percent divided of CD4 + CD3 + CPD + T cells. Asterisk indicates a significant difference, ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001. Significance was determined by Kruskal-Wallis test (C–E and G), (F) Mann-Whitney U test, and (I) Signed Wilcoxon-rank test. Data are represented as mean ± SEM (D and I) or median (E, F, H, and I) are shown.

Article Snippet: APC anti-human CD25 (clone BC96) , eBioscience , Cat#17-0259-42; RRID: AB_1582219.

Techniques: In Vitro, Cell Isolation, Staining, Expressing, Labeling, Comparison, Co-Culture Assay, Cell Culture, Generated, MANN-WHITNEY

Journal: iScience

Article Title: Single cell profiling of hematopoietic stem cell transplant recipients reveals TGF-β1 and IL-2 confer immunoregulatory functions to NK cells

doi: 10.1016/j.isci.2024.111416

Figure Lengend Snippet:

Article Snippet: APC anti-human CD25 (clone BC96) , eBioscience , Cat#17-0259-42; RRID: AB_1582219.

Techniques: Recombinant, Staining, Selection, Software

Journal: PLoS ONE

Article Title: Dual Role of miR-21 in CD4+ T-Cells: Activation-Induced miR-21 Supports Survival of Memory T-Cells and Regulates CCR7 Expression in Naive T-Cells

doi: 10.1371/journal.pone.0076217

Figure Lengend Snippet: A Resting naive (CD3+ CD8-CD45RO-CD25-) T-cells were stimulated with 5µg/ml PHA and 100 U/mL IL-2, followed by transduction with lentiviral vectors harboring miR-21 or control inhibitor (scrambled hairpin sequence) and GFP. The percentage of GFP+ cells in culture over time was monitored by FACS. Data were normalized to the first measurement at day six. Each line represents a separate donor ( n =5, two-way RM ANOVA with Bonferroni posttests, ns-not significant). B Data from A, depicted as median values with interquartile range. C Resting memory (CD3+ CD8-CD45RO-CD25-) T-cells were stimulated with 5µg/ml PHA and 100 U/mL IL-2, followed by transduction with lentiviral vectors harboring miR-21 or control inhibitor (scrambled hairpin sequence) and GFP. The percentage of GFP+ cells in culture over time was monitored by FACS. Data were normalized to the first measurement at day six. Each line represents a separate donor ( n =5, two-way RM ANOVA with Bonferroni posttests). D Data from C, depicted as median with interquartile range. E Activated naive, and F activated memory GFP+ T-cells harboring miR-21 or control inhibitor were isolated by FACS from mixed cultures (A, and C respectively) at day six post lentiviral transduction. Isolated GFP+ cells, and activated not transduced cells (NT) were cultured in complete media supplemented with 100 U/mL of IL-2 for 48h (until day 8), and percentage of apoptotic cells was assessed by FACS-based measurement of mitochondrial trans-membrane potential loss, using DilC1(5). Each line represents a separate donor (naive: n =4, memory n=5 , RM ANOVA with Bonferroni posttests). *p<0.05, **p<0.01, ***p<0.0001.

Article Snippet: Expression of cell-surface markers on T-cell subsets before and after treatment with activating stimuli was assessed by FACS using mAbs against human CD45RO-PE (UCHL1), CD25-APC (BC96), CD45RA-eFluor605 (HI100, all from eBioscience), and CCR7-PE-Cy7 (3D12) or rat-IgG2a-PE-Cy7 isotype control (R35-9, both from BD Biosciences).

Techniques: Transduction, Sequencing, Isolation, Cell Culture

Journal: PLoS ONE

Article Title: Dual Role of miR-21 in CD4+ T-Cells: Activation-Induced miR-21 Supports Survival of Memory T-Cells and Regulates CCR7 Expression in Naive T-Cells

doi: 10.1371/journal.pone.0076217

Figure Lengend Snippet: A Representative FACS staining plot depicting CCR7 expression on resting naive (CD3+ CD8-CD45RO-CD25-, Day 0), and on PHA/IL-2 activated, GFP+ cells harboring miR-21 or control inhibitor (scrambled hairpin sequence) at day six post lentiviral transduction. The isotype staining control is depicted. B Quantification of CCR7 staining of GFP+ cells from A, depicted as MFI (geometric mean fluorescent intensity). Each line represents a separate donor ( n=8 , Wilcoxon signed ranked test). C Percentage of CCR7 positive, GFP+ cells from A. Each line represents a separate donor ( n=8 , Wilcoxon signed ranked test). D GFP+, PHA/IL-2 activated naive T-cells harboring miR-21 or control inhibitor were FACS isolated at day six post-lentiviral transduction and CCR7 transcript expression was determined by qRT-PCR. Each line represents a separate donor ( n=3 , paired t-test, ns). Relative expression normalized to the U6 reference gene is shown. E Representative FACS staining plot depicting CCR7 expression on resting memory (CD3+ CD8-CD45RO+CD25-, Day 0), and on PHA/IL-2 activated, GFP+ cells harboring miR-21 or control inhibitor (scrambled hairpin sequence) at day six post lentiviral transduction. The isotype staining control is also depicted. F Quantification of CCR7 staining of GFP+ cells from E, depicted as MFI (geometric mean fluorescent intensity). Each line represents a separate donor ( n=7 , Wilcoxon signed ranked test). G Percentage of CCR7 positive, GFP+ cells from E. Each line represents a separate donor ( n=7 , Wilcoxon signed ranked test). D GFP+, PHA/IL-2 activated memory T-cells harboring miR-21 or control inhibitor were FACS isolated at day six post-lentiviral transduction and CCR7 transcript expression was determined by qRT-PCR. Each line represents a separate donor ( n=3 , paired- t-test, ns). Relative expression normalized to the U6 reference gene is shown. *p<0.05, **p<0.01.

Article Snippet: Expression of cell-surface markers on T-cell subsets before and after treatment with activating stimuli was assessed by FACS using mAbs against human CD45RO-PE (UCHL1), CD25-APC (BC96), CD45RA-eFluor605 (HI100, all from eBioscience), and CCR7-PE-Cy7 (3D12) or rat-IgG2a-PE-Cy7 isotype control (R35-9, both from BD Biosciences).

Techniques: Staining, Expressing, Sequencing, Transduction, Isolation, Quantitative RT-PCR

Journal: Medicine

Article Title: Intramuscular administration of autologous total immunoglobulin G induces immunomodulatory effects on T cells in healthy human subjects

doi: 10.1097/MD.0000000000029486

Figure Lengend Snippet: The percentages of IL-10- or IFN-γ-producing peripheral blood CD3 + (A), CD4 + (B), CD8 + (C), and CD4 + CD25 + (D) T cells in 13 healthy human subjects who received 8 intramuscular administrations of 50 mg autologous total IgG twice a week for 4 wk (from week 0 to week 4) during the 12-wk study period. P values were calculated using the Wilcoxon signed-rank test with Bonferroni correction. Each symbol and connecting line in the graphs represents an individual healthy subject. ∗ P < .05, ∗∗ P < .01 compared to baseline.

Article Snippet: Next, cells were incubated with PerCP-Cyanine5.5-labeled anti-CD3 (SK7, eBioscience), FITC-labeled anti-CD4 (RPA-T4, eBioscience), PE-labeled anti-CD4 (RPA-T4, eBioscience), PE-Cyanine7-labeled anti-CD8 (SK1, eBioscience), APC-labeled anti-CD25 (BC96, eBioscience), and PE-Cyanine7-labeled anti-CD19 (HIB19, eBioscience) antibodies for 30 minutes at 4 °C.

Techniques:

Journal: Antioxidants

Article Title: Role of Oxidative Stress in Vascular Low-Grade Inflammation Initiation Due to Acute Salt Loading in Young Healthy Individuals

doi: 10.3390/antiox11030444

Figure Lengend Snippet: Gating strategy for the assessment of peripheral blood regulatory T cells (Treg) by flow cytometry. Panel ( A ) shows representative dot plots illustrating gating strategy, including exclusion of doublets using forward scatter area (FSC-A) versus forward scatter width (FSC-W) analysis ( A - i ), gating on live cells negative for aminereactive fixable viability dye ( A - ii ), lymphocytes ( A - iii ), and CD3 + T cells ( A - iv ). Next, total CD25 and Foxp3, expressing T cells among CD4 + CD127 low population, were analyzed (Panel ( B )). ( B - i ) shows representative gating strategy, ( B - ii ) representative samples from LS and HS protocol. FlowLogic software was used for data analysis and illustration.

Article Snippet: In order to determine expression of Treg specific cell surface antigens, the following antibody mixture was used: CD3 FITC (clone: OKT3, eBioscienceTM, Affymetrix by Thermo Fisher Scientific, CA, USA), CD4 PerCP-eFluorTM 710 (clone: SK3, eBioscienceTM), CD127 PE-Cy7 (clone: eBioRDR5, eBioscienceTM), and CD25 APC (clone: BC96, eBioscienceTM); the Foxp3 PE (clone: 235A/E7, eBioscienceTM) antibody was used for the detection of Treg signature transcription factor Foxp3.

Techniques: Flow Cytometry, Expressing, Software