Journal:

Article Title: Factors affecting the susceptibility of the mouse pituitary gland to CD8 T-cell-mediated autoimmunity

doi: 10.1111/j.1365-2567.2004.01821.x

Figure Lengend Snippet: (a) In vitro proliferation of carboxy fluorescein diacetate succinimidyl ester (CFSE)-labelled F5 T cells in response to cross-presentation of Δ.2-Rag−/−, 48-Rag−/− and Rag−/− pituitary extracts by bone marrow-derived H-2b dendritic cells (DCs). (b) In vitro proliferation of CFSE-labelled F5 T cells incubated with DCs and 1 nm ASNENMDAM peptide. (c) Semi-quantitative PCR analysis of influenza nucleoprotein A/NT/60/68 (NP) transgene expression. RNA was extracted from the pituitaries of 48-Rag−/− and D.2-Rag−/− mice and reverse transcribed, as described. Dilutions of cDNA were amplified with primers specific to NP and growth hormone (GH). The GH semiquantitative PCR was used to control for the concentration of cDNA in each sample.

Article Snippet: F5 lymph node T cells were incubated at 37° for 10 min at a cell density of 10 7 /ml with 1 μ m carboxy fluorescein diacetate succinimidyl ester (CFSE) (Molecular Probes, Eugene, OR) in phosphate-buffered saline (PBS) and washed thoroughly.

Techniques: In Vitro, Derivative Assay, Incubation, Real-time Polymerase Chain Reaction, Expressing, Reverse Transcription, Amplification, Control, Concentration Assay

Journal:

Article Title: Factors affecting the susceptibility of the mouse pituitary gland to CD8 T-cell-mediated autoimmunity

doi: 10.1111/j.1365-2567.2004.01821.x

Figure Lengend Snippet: Characteristics of double-transgenic Δ.2-Rag−/− F5 mice. (a) Growth of male Δ.2-Rag−/− F5 (•) mice compared with F5 (○) and 48-Rag−/− F5 (×) mice. Growth data for the 48-Rag−/− F5 mice have been previously published20 and are included here for comparison only. (b) Pituitary GH levels in individual 16-week-old male Δ.2-Rag−/− F5 (•; n = 4) and Rag−/− F5 (○; n = 6) mice. The mean value for each group is indicated by a horizontal line. (c) High CD44 expression in pituitary-infiltrating CD8+ T-cell receptor (TCR)+ cells isolated from a 12-week-old Δ.2-Rag−/− F5 mouse. (d) In vivo proliferation of transferred, CFSE-labelled F5 T cells in the cervical lymph nodes of B10, 48-B10 and Δ.2-B10 mice of various ages. B10 mice (n = 5), 48-B10 (13 days old; n = 8) and Δ.2-B10 mice (14 days old, n = 7; 23 days old, n = 5; and > 35 days old, n = 10) were injected intravenously (i.v.) with 10 × 106 T cells. The percentage of transferred T cells that had undergone one or more divisions was determined by fluorescence-activated cell sorter (FACS) analysis, 4 days after cell transfer. Numbers indicate the proportion of mice in each group in which the F5 T-cell proliferation was observed.

Article Snippet: F5 lymph node T cells were incubated at 37° for 10 min at a cell density of 10 7 /ml with 1 μ m carboxy fluorescein diacetate succinimidyl ester (CFSE) (Molecular Probes, Eugene, OR) in phosphate-buffered saline (PBS) and washed thoroughly.

Techniques: Transgenic Assay, Comparison, Expressing, Isolation, In Vivo, Injection, Fluorescence

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: CD45RA − Foxp3 high regulatory T cells have a negative impact on the clinical outcome of head and neck squamous cell carcinoma

doi: 10.1007/s00262-017-2021-z

Figure Lengend Snippet: Frequency and characteristics of Treg subsets in patients with HNSCC and in those with benign tumors. The gating strategies for Treg subsets in the PBMCs of patients with HNSCC or benign tumors. I: CD45RA+Foxp3low, II: CD45RA−Foxp3low, and III: CD45RA−Foxp3high (a). The expression of CTLA-4, CD25, and CCR4 on Treg subsets in a representative patient with HNSCC (b). Mean fluorescence intensities (MFI) are indicated in the upper right quadrant. CFSE dilutions of proliferated CD4+ T cells were assessed after 72 h of TCR stimulation with or without indicated Treg subsets at a 1:1 ratio (c), and the number of proliferated CD4+ T cells after the co-culture are shown (d). The percentage of Foxp3+ cells in CD4+ T cells (e), CD45RA−Foxp3high Tregs in CD4+ T cells (F), CD45RA+Foxp3low Tregs in CD4+ T cells (g) and the ratio of CD45RA−Foxp3high Tregs to CD45RA+Foxp3low Tregs (h) in PBMCs of patients with HNSCC (n = 46) and benign tumors (n = 23) before treatment. The percentage of CD45RA−Foxp3high Tregs and the ratio of CD45RA−Foxp3high Tregs to CD45RA+Foxp3low Tregs were increased according to clinical stage (i). Statistical comparisons were performed using the parametric unpaired t test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. n.s. not significant, HNSCC head and neck squamous cell carcinoma, CTLA-4 cytotoxic T-lymphocyte antigen 4, CCR CC chemokine receptor, CFSE carboxyfluorescein diacetate succinimidyl ester, TCR T cell receptor

Article Snippet: Enriched CD3 + T cells were labeled with CellTrace CFSE (carboxyfluorescein diacetate succinimidyl ester) Cell Proliferation Kit (Molecular Probes, USA) according to the manufacturer’s instructions.

Techniques: Expressing, Fluorescence, Co-Culture Assay

Journal: Chinese Journal of Cancer

Article Title: Ex vivo expansion of tumor-infiltrating lymphocytes from nasopharyngeal carcinoma patients for adoptive immunotherapy

doi: 10.5732/cjc.011.10376

Figure Lengend Snippet: A, representative experiments illustrate the EBV-specific cytotoxic activity of TILs from three different NPC patients to target a panel of cells, including auto-LCL (EBV + ), auto-blast cells (EBV − ), NPC tumor cell line C666 (EBV + ), and leukemia cell line K562 (EBV − ). The effector cell and target cell panels were co-cultured at different ratios for 5 h, and target cells were labeled by 5- or 6-(N-Succinimidyloxycarbonyl)-3′,6′-O,O′-diacetylfluorescein (CFSE). The percentage of specific lysis of target cells was determined by 7-amino-actinomycin D (7-AAD) staining and fluorescence-activated cell sorting (FACS) detection. One of three experiments is shown in the figure. B, the average percentage of specific lysis of TILs to target cells, including auto-LCLs and auto-blast cells ( n = 15), is shown in the figure. Error bars represent standard deviation.

Article Snippet: The target cells were labeled with 5- or 6-(N-Succinimidyloxycarbonyl)-3′, 6′-0,0′'- diacetylfluorescein (CFSE; Molecular Probes™, Invitrogen, Paisley, UK), co-cultured with TILs for 5 h, and then harvested and stained with 7-amino-actinomycin D (7-AAD) (eBioScience) to detect dead cells.

Techniques: Activity Assay, Cell Culture, Labeling, Lysis, Staining, Fluorescence, FACS, Standard Deviation

Journal: Advanced Science

Article Title: A Temporal PROTAC Cocktail‐Mediated Sequential Degradation of AURKA Abrogates Acute Myeloid Leukemia Stem Cells

doi: 10.1002/advs.202104823

Figure Lengend Snippet: Characterization of cellular response to PROTACs in AML cells. A) KG1A and Kasumi‐1 cells were stained with a carboxyfluorescein succinimidyl amino ester (CFSE) probe and cultured with AURKA PROTACs (1 × 10 −6 m ) for 72 h. CFSE fluorescence was analyzed by flow cytometry. MFI, mean fluorescence intensity. B) KG1A cells were treated with AURKA PROTACs (1 × 10 −6 m ) for 48 h. The cell cycle profile was assayed by FACS with propidium iodide (PI) staining. The cell cycle phase distribution was analyzed by FlowJo software. C) A heatmap of the relative normalized abundance of proteins in TMT‐based quantitative proteomic assays. D) TMT‐based quantitative proteomics after treatment with PROTACs (500 × 10 −9 m ) or the DMSO Vehicle for 6 h in KG1A cells. The top 100 decreased proteins were subjected to g:Profiler to perform gene ontology (GO) analysis. E) Gene set enrichment analysis (GSEA) of RNA‐seq data from KG1A cells treated with AURKA PROTACs (1 × 10 −6 m ) for 48 h. F) KG1A cells were treated with AURKA PROTACs (1 × 10 −6 m ) or ATRA (0.6 × 10 −6 m ) for 48 h. Cell surface CD34 expression was analyzed by FACS. MFI, mean fluorescence intensity. G) KG1A cells were arrested with nocodazole (0.1 µg mL −1 ) for 16 h followed by PROTACs or MLN8237 treatment with indicated concentration for 6 h. AURKA localization were detected by immunofluorescence. Scale bar: 5 µm. H) KG1A cells were arrested with nocodazole (0.1 µg mL −1 ) for 16 h followed by DMSO, PROTACs (1 × 10 −6 m ) or MLN8237 (50 × 10 −9 m ) treatment for indicated times. The expression of AURKA and related proteins were detected. Statistics, significance: one‐way ANOVA with Bonferroni correction (A,F); ns, not significant; * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: Briefly, cells were prestained with CFSE probe (S8269, Selleck, 0.5 × 10 −6 m ) in a serum free medium for 15 min, washed with PBS and then suspended and cultured in fresh medium with indicated concentrations of AURKA PROTACs for 72 h. Then, cells were collected, washed, and resuspended in 250 μL PBS.

Techniques: Staining, Cell Culture, Fluorescence, Flow Cytometry, Software, Quantitative Proteomics, RNA Sequencing, Expressing, Concentration Assay, Immunofluorescence

Journal: Advanced Science

Article Title: A Temporal PROTAC Cocktail‐Mediated Sequential Degradation of AURKA Abrogates Acute Myeloid Leukemia Stem Cells

doi: 10.1002/advs.202104823

Figure Lengend Snippet: dAurA383 and dAurA450 synergistically inhibit the growth and stemness of AML cells. A,B) KG1A and Kasumi‐1 cells were treated with various concentrations of dAurA383, dAurA450 alone or in combination for 72 h. The growth inhibitory effects were determined by CCK8 staining. The CI value was calculated with CompuSyn software. C) KG1A and Kasumi‐1 cells were stained with carboxyfluorescein succinimidyl amino ester (CFSE) probe and cultured with PROTACs (500 × 10 −9 m ) for 72 h. CFSE fluorescence was analyzed by flow cytometry. MFI, mean fluorescence intensity. D) KG1A and Kasumi‐1 cells were treated with AURKA PROTACs (500 × 10 −9 m ) for 48 h. The cell cycle profile was assayed by FACS with propidium iodide (PI) staining. The cell cycle phase distribution was analyzed by FlowJo software. E) The apoptosis of KG1A and Kasumi‐1 cells treated with the indicated PROTACs (500 × 10 −9 m ) for 48 h. The rate of Annexin V positive cells was calculated. F) KG1A cells were treated with AURKA PROTACs (500 × 10 −9 m ) for 48 h. Cell surface CD34 expression was analyzed by FACS. MFI, mean fluorescence intensity. G) Methylcellulose‐based colony forming cell (CFC) assays to examine the stemness of KG1A cells treated with PROTACs (500 × 10 −9 m ) for 14 days. Sphere number (diameter > 50 µm, left panel) and size (diameter, right panel) were calculated. Scale bar: 50 µm. H,I) KG1A cells were treated with AURKA PROTACs (500 × 10 −9 m ) for 48 h. ALDH positive cells were analyzed by flow cytometry assays. J&K. KG1A and Kasumi‐1 cells were treated with AURKA PROTACs (1 × 10 −6 m ) and MLN8237 (50 × 10 −9 m ) for 48 h. Western blot analysis of AURKA as well as stemness markers c‐Myc, NANOG, STAT5A, and FOXM1. Statistics, significance: one‐way ANOVA with Bonferroni correction (A,C,E,F,G,I); ns, not significant; * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: Briefly, cells were prestained with CFSE probe (S8269, Selleck, 0.5 × 10 −6 m ) in a serum free medium for 15 min, washed with PBS and then suspended and cultured in fresh medium with indicated concentrations of AURKA PROTACs for 72 h. Then, cells were collected, washed, and resuspended in 250 μL PBS.

Techniques: Staining, Software, Cell Culture, Fluorescence, Flow Cytometry, Expressing, Western Blot

Journal: Clinical Cancer Research

Article Title: Effective Photoimmunotherapy of Murine Colon Carcinoma Induced by the Combination of Photodynamic Therapy and Dendritic Cells

doi: 10.1158/1078-0432.ccr-04-0367

Figure Lengend Snippet: Fig. 3 Capture of photodynamic therapy (PDT)-treated C-26 cells by immature dendritic cells (DCs). C-26 cells were labeled with a fluorescent dye [5,6-carboxyfluorescein diacetate succinimidyl ester(CFSE)] before incubation with Photofrin (5 g/ml) and illumination with laser light (10 kJ/m2). DCs were added to PDT-treated C-26 cells 4 h after PDT and were incubated for another 4 h before staining with PE-conjugated antimouse I-A/I-E monoclonal antibody against MHC class II antigen. Confocal laser scanning microscopy in A, DCs (red) interacting with CFSE-labeled C-26 cells (green, A1) as well as fragments of C-26 cells ingested by DCs (A2, A3). B and D, dot-plots of cells showing CFSE fluorescence; these cells were gated for the expression of MHC class II molecules. C, CFSE-labeled C-26 cells. E, a histogram composition of cells in B–D, showing a relative CFSE fluorescence in DCs (red), DCs cocultured with CFSE-labeled C-26 cells (orange), and CFSE-labeled C-26 cells (green). F, the concentrations of interleukin 12 (IL-12) secreted by DCs cocultured with PDT-treated C-26 cells. C-26 cells were plated into wells of 96-well plates (2 104/well) and were incubated for 24 h with Photofrin (5 mg/ml). After illumination with a laser light (10 kJ/m2), the cells were incubated for 4 h at 37°C and 5% CO2. Then, immature bone marrow-derived DCs (1 105) were added and cocultured with PDT-treated C-26 cells for another 24 h. The concentrations of IL-12 were measured directly from culture supernatants. B–E, SSC, side scatter.

Article Snippet: A vital nonphototoxic fluorescent dye, 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE), was purchased from Molecular Probes (Leiden, the Netherlands).

Techniques: Labeling, Incubation, Staining, Confocal Laser Scanning Microscopy, Fluorescence, Expressing, Derivative Assay

Journal:

Article Title: Simian Immunodeficiency Virus Nef Protein Delays the Progression of CD4 + T Cells through G 1 /S Phase of the Cell Cycle

doi: 10.1128/JVI.76.8.3587-3595.2002

Figure Lengend Snippet: Analysis of the effects of Nef on cell proliferation. Jurkat, B5 uninduced, and B5 induced cells were labeled with CFSE and cultured for 5 days. Cells were harvested, and fluorescence intensity and cell counts were analyzed by flow cytometry. Proliferation and cell division were characterized by sequential reduction in CFSE fluorescence intensity, generating a series of shifted equally spaced peaks on a logarithmic scale. (A) Comparison of CFSE intensity profiles of freshly labeled Jurkat cells (left) and cells after 5 days of culture (right). (B) Histograms showing different discrete cycles of cell division. Shown are cell division cycles of Jurkat and B5 uninduced cells (top) and B5 induced cells (bottom) after 5 days of culture.

Article Snippet: To determine the effect of Nef on cell proliferation, Jurkat cells, uninduced and induced B5 cells, were labeled with 5- and 6-carboxyfluorescein diacetate succinimidyl ester (CFSE; Molecular Probes Inc., Eugene, Oreg.) as previously described ( 33 ).

Techniques: Labeling, Cell Culture, Fluorescence, Flow Cytometry