phycoerythrin pe conjugated anti cd69  (Thermo Fisher)

Journal: PLoS ONE

Article Title: HIV-Induced Type I Interferon and Tryptophan Catabolism Drive T Cell Dysfunction Despite Phenotypic Activation

doi: 10.1371/journal.pone.0002961

Figure Lengend Snippet: PBMC from HIV-uninfected donors were cultured for 24 and 48 hours in presence of control microvescicles, HIV alone or in presence of blocking antibodies against the cellular receptor for IFN-α (anti-IFNAR). CD38 and CD69 expression were analyzed by flow cytometry on gated CD3 + CD4 + and CD3 + CD8 + cells (CD4 and CD8 T cells, respectively). (A) and (C) show flow cytometry contour plots of CD69 and CD38 expression for one example experiment for CD4 and CD8 T cells, respectively. (B) and (D) show bar graphs summarizing mean fluorescence intensity (MFI) of CD38 and CD69 in CD4 and CD8 T cells, respectively (48 hours only). Mean values±standard error calculated on 5 independent experiments are shown in the bar graphs.

Article Snippet: After stimulation in culture, cells were washed and incubated for 20 min at room temperature in PBS containing 2% mouse serum (Sigma) with the following antibodies: Peridinn chlorophyll protein (PerCP)-conjugated anti-CD3 (BD Biosciences), Allophycocyanin (APC)-conjugated anti-CD4 (BD Biosciences), PE-Cy7-conjugated anti-CD8 (BD Biosciences), Phycoerythrin (PE)-conjugated anti-CD69 (BD Biosciences), fluorescein isothiocyanate (FITC)-conjugated anti-CD38 (BD Biosciences).

Techniques: Cell Culture, Blocking Assay, Expressing, Flow Cytometry, Fluorescence

Journal: PLoS ONE

Article Title: HIV-Induced Type I Interferon and Tryptophan Catabolism Drive T Cell Dysfunction Despite Phenotypic Activation

doi: 10.1371/journal.pone.0002961

Figure Lengend Snippet: PBMC from HIV-uninfected donors were cultured for 24 (upper panels) and 48 hours (lower panels) in presence or absence of recombinant IFN-α (rIFN-α). CD38 and CD69 expression were analyzed by flow cytometry on gated CD3 + CD4 + and CD3 + CD8 + cells (CD4 and CD8 T cells, respectively). Flow cytometry contour plots of CD69 and CD38 expression for one example experiment for CD4 (left panels) and CD8 T cells (right panels).

Article Snippet: After stimulation in culture, cells were washed and incubated for 20 min at room temperature in PBS containing 2% mouse serum (Sigma) with the following antibodies: Peridinn chlorophyll protein (PerCP)-conjugated anti-CD3 (BD Biosciences), Allophycocyanin (APC)-conjugated anti-CD4 (BD Biosciences), PE-Cy7-conjugated anti-CD8 (BD Biosciences), Phycoerythrin (PE)-conjugated anti-CD69 (BD Biosciences), fluorescein isothiocyanate (FITC)-conjugated anti-CD38 (BD Biosciences).

Techniques: Cell Culture, Recombinant, Expressing, Flow Cytometry

Journal: Experimental and Therapeutic Medicine

Article Title: Effects of CD4 + T lymphocytes from ovariectomized mice on bone marrow mesenchymal stem cell proliferation and osteogenic differentiation

doi: 10.3892/etm.2020.9212

Figure Lengend Snippet: CD4 + T cells are activated in OVX mice. (A) The gating strategies used for CD4 + T cells. (B) Percentage of CD4 + CD69 + cells in spleens tested using flow cytometry. (C) Expression levels of proinflammatory cytokines, IL-2, IFN-γ and TNF-α, in purified CD4 + T cells were determined using reverse transcription-quantitative-PCR. (D) TNF-α levels in the supernatant from purified CD4 + T cell cultures were measured by ELISA. * P<0.05, ** P<0.01 vs. sham. CD, cluster of differentiation; OVX, ovariectomized; IL-2, interleukin 2; IFN-γ, interferon- γ; TNF-α, tumor necrosis factor-α; IgG, immunoglobulin; APC, allophycocyanin.

Article Snippet: Inc.) and magnetic beads coated with anti-mouse CD4 antibodies (Miltenyi Biotec., Inc.), CD3 and CD28 (both, BD Biosciences), a volumetric microscope, an inverted phase contrast microscope and camera system (Olympus Corporation), a flow cytometer (Beckman Coulter, Inc.), an Alkaline Phosphatase Staining kit (cat. no. P0321; Beyotime Institute of Biotechnology), a mouse TNF-α ELISA kit (cat. no. MTA00B; R&D Systems, Inc.), anti-TNF-α antibodies (cat. no. AF-410-NA; R&D Systems, Inc.), FITC-conjugated anti-CD3 (cat. no. 11-0032-82; eBioscience; Thermo Fisher Scientific, Inc.), allophycocyanin (APC)-conjugated anti-CD4 (cat. no. 17-0042-82; eBioscience; Thermo Fisher Scientific, Inc.), phycoerythrin (PE)-conjugated anti-CD69 (cat. no. 12-0691-82; eBioscience; Thermo Fisher Scientific, Inc.) and Armenian hamster IgG isotype control (cat. no. 12-4888-81; eBioscience; Thermo Fisher Scientific, Inc.).

Techniques: Flow Cytometry, Expressing, Purification, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: PTPN3 expressed in activated T lymphocytes is a candidate for a non-antibody-type immune checkpoint inhibitor

doi: 10.1007/s00262-019-02403-y

Figure Lengend Snippet: Lymphocyte activation by PTPN3 inhibition was observed only in CD3+ T cells, not in CD3− cells. a An FACS analysis of activated lymphocytes before selection. CD3+CD4+NKG2D− cells (b) and CD3+CD8+NKG2D+ cells (c) were separately collected using microbeads. CD3− (NK) cells (d) were collected using FACS sorting. e The migration ability of lymphocytes was analyzed by time-lapse imaging. The graph shows the moving distance of lymphocytes analyzed by the Image-Pro Analyzer software program. f The proliferation of FAR red-labeled lymphocytes was estimated by FACS. g The PTPN3 mRNA expression in CD3− cells and CD3+ cells after activation was estimated by real-time RT-PCR. h The migration ability of non-activated lymphocytes was analyzed by the chamber method. i The cytotoxicity of non-activated lymphocytes was investigated. After co-culture of SCC cells and lymphocytes for 72 h, the absorbance of viable cancer cells was measured. j The PTPN3 mRNA expression in lymphocytes stimulated by allo-pancreatic cancer (SUIT-2) cell-pulsed mature DCs without using anti CD3 mAb was estimated by real-time RT-PCR. Similar results were obtained in two different healthy volunteers in all experiments. Data are presented as the mean ± SD. Lymphocytes and DCs from healthy volunteers were used in all experiments. *p < 0.05

Article Snippet: Fluorescence-activated cell sorting (FACS) Cells were stained with fluorescein isothiocyanate (FITC)-conjugated anti-CD3, CD4, and monoclonal antibodies (mAbs), or phycoerythrin (PE)-conjugated anti-NKG2D, CD8 and CD69 mAbs (BD Pharmingen, San Diego, CA, USA).

Techniques: Activation Assay, Inhibition, Selection, Migration, Imaging, Software, Labeling, Expressing, Quantitative RT-PCR, Co-Culture Assay

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: PTPN3 expressed in activated T lymphocytes is a candidate for a non-antibody-type immune checkpoint inhibitor

doi: 10.1007/s00262-019-02403-y

Figure Lengend Snippet: PTPN3 inhibition increases the cytotoxicity against cancer cells in vivo. a–f Therapy experiments using allo-activated lymphocytes targeting SUIT-2 cells from healthy volunteers are shown. Allo-activated lymphocytes transfected with control shRNA or PTPN3#2 shRNA were administered at 6, 9, 13, and 16 days after tumor injection (arrows in b). a Representative photos of formed tumors. b The tumor volume at the indicated days and c Kaplan–Meier plot of the tumor growth are shown. d The tumor weight at 22 days after tumor injection was assessed. e Representative photos of immunohistochemical staining of CD8 in formed tumors. Original magnification ×200. f The numbers of labeled tumor-infiltrating-administrated lymphocytes (TILs) were counted. g–l Therapy experiments using tumor and autologous lymphocytes from a patient with ovarian cancer are shown. Autologous-activated lymphocytes transfected with control shRNA or PTPN3#2 shRNA were administered at 11, 14, 18, and 21 days after tumor injection (arrows in h). g Representative photos of formed tumors. h The tumor volume at the indicated days and i Kaplan–Meier plot of the tumor growth are shown. j Representative photos of immunohistochemical staining of CD3 in formed tumors. Original magnification ×200. k The numbers of non-tumor-tissue-infiltrating-administrated lymphocytes (ntTILs) were counted. l The numbers of ntTILs in the spleen, liver and lung were counted. Data are presented as the mean ± SD. *p < 0.05

Article Snippet: Fluorescence-activated cell sorting (FACS) Cells were stained with fluorescein isothiocyanate (FITC)-conjugated anti-CD3, CD4, and monoclonal antibodies (mAbs), or phycoerythrin (PE)-conjugated anti-NKG2D, CD8 and CD69 mAbs (BD Pharmingen, San Diego, CA, USA).

Techniques: Inhibition, In Vivo, Transfection, shRNA, Injection, Immunohistochemical staining, Staining, Labeling

Journal: eLife

Article Title: Calcium-mediated shaping of naive CD4 T-cell phenotype and function

doi: 10.7554/eLife.27215

Figure Lengend Snippet:

Article Snippet: Antibody , Phycoerythrin (PE)-conjugated anti-CD69 (H1.2F3) , BD Biosciences , Cat# 553237.

Techniques: Recombinant, Software, Expressing