peripheral blood mononuclear cells pbmcs  (Valiant)

 
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    Valiant peripheral blood mononuclear cells pbmcs
    Isolation and molecular characterization of T cell subsets from <t>PNH</t> patients ( A ) Gating strategy for T cell subsets for cell sorting. <t>PBMCs</t> were stained with ViViD, anti-CD14-Pacific Blue, anti-CD19-Pacific Blue, anti-CD3-BV605, anti-CD4-V500, anti-CD8-APC-H7, anti-CD45RA-PE-Cy7, and anti-CD45RO-APC. Lymphocytes were gated based on their scatter characteristics or forward scatter height vs. forward scatter area. Live T cells were gated based on positive for CD3 and negative for ViViD, CD14, and CD19 to remove dead cells, monocytes, and B cells. CD4 + and CD8 + T cells were then gated based on the characteristic expression patterns of CD45RA and CD45RO. Four different T cell subsets [CD4 + naïve (CD45RA + CD45RO - ), CD4 + memory (CD45RA - CD45RO + ), CD8 + naïve (CD45RA + CD45RO - ), and CD8 + memory (CD45RA - CD45RO + ) T cells] were sorted for RNA-seq. ( B ) Transcriptional validation of the naïve and memory T cell identity by analyzing expression levels of CD4, CD8, CCR5, and EOMES. FPKM (fragments per kilobase of exon per million fragments mapped). * p
    Peripheral Blood Mononuclear Cells Pbmcs, supplied by Valiant, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "T Cell Transcriptomes from Paroxysmal Nocturnal Hemoglobinuria Patients Reveal Novel Signaling Pathways"

    Article Title: T Cell Transcriptomes from Paroxysmal Nocturnal Hemoglobinuria Patients Reveal Novel Signaling Pathways

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    doi: 10.4049/jimmunol.1601299

    Isolation and molecular characterization of T cell subsets from PNH patients ( A ) Gating strategy for T cell subsets for cell sorting. PBMCs were stained with ViViD, anti-CD14-Pacific Blue, anti-CD19-Pacific Blue, anti-CD3-BV605, anti-CD4-V500, anti-CD8-APC-H7, anti-CD45RA-PE-Cy7, and anti-CD45RO-APC. Lymphocytes were gated based on their scatter characteristics or forward scatter height vs. forward scatter area. Live T cells were gated based on positive for CD3 and negative for ViViD, CD14, and CD19 to remove dead cells, monocytes, and B cells. CD4 + and CD8 + T cells were then gated based on the characteristic expression patterns of CD45RA and CD45RO. Four different T cell subsets [CD4 + naïve (CD45RA + CD45RO - ), CD4 + memory (CD45RA - CD45RO + ), CD8 + naïve (CD45RA + CD45RO - ), and CD8 + memory (CD45RA - CD45RO + ) T cells] were sorted for RNA-seq. ( B ) Transcriptional validation of the naïve and memory T cell identity by analyzing expression levels of CD4, CD8, CCR5, and EOMES. FPKM (fragments per kilobase of exon per million fragments mapped). * p
    Figure Legend Snippet: Isolation and molecular characterization of T cell subsets from PNH patients ( A ) Gating strategy for T cell subsets for cell sorting. PBMCs were stained with ViViD, anti-CD14-Pacific Blue, anti-CD19-Pacific Blue, anti-CD3-BV605, anti-CD4-V500, anti-CD8-APC-H7, anti-CD45RA-PE-Cy7, and anti-CD45RO-APC. Lymphocytes were gated based on their scatter characteristics or forward scatter height vs. forward scatter area. Live T cells were gated based on positive for CD3 and negative for ViViD, CD14, and CD19 to remove dead cells, monocytes, and B cells. CD4 + and CD8 + T cells were then gated based on the characteristic expression patterns of CD45RA and CD45RO. Four different T cell subsets [CD4 + naïve (CD45RA + CD45RO - ), CD4 + memory (CD45RA - CD45RO + ), CD8 + naïve (CD45RA + CD45RO - ), and CD8 + memory (CD45RA - CD45RO + ) T cells] were sorted for RNA-seq. ( B ) Transcriptional validation of the naïve and memory T cell identity by analyzing expression levels of CD4, CD8, CCR5, and EOMES. FPKM (fragments per kilobase of exon per million fragments mapped). * p

    Techniques Used: Isolation, FACS, Staining, Expressing, RNA Sequencing Assay

    2) Product Images from "Tumor-derived exosomes educate dendritic cells to promote tumor metastasis via HSP72/HSP105-TLR2/TLR4 pathway"

    Article Title: Tumor-derived exosomes educate dendritic cells to promote tumor metastasis via HSP72/HSP105-TLR2/TLR4 pathway

    Journal: Oncoimmunology

    doi: 10.1080/2162402X.2017.1362527

    Exosomes from tumor patients with increased membrane-associated HSP72 and HSP105 induced DCs to promote tumor cell invasion via IL-6 in vitro . (A) After adsorption onto latex beads, HSP72 and HSP105 on EXO-PT and EXO-TT were detected by flow cytometry. RFI was calculated by dividing mean fluorescence intensity of samples staining with detection Abs by that of sample staining with ISO Abs (n = 7). (B) Human DCs were induced from PBMCs and stimulated with 5 μg/ml of EXO-PT or EXO-TT from breast tumor tissues for 6 h. The IL-6 production was measured by ELISA (n = 3). (C) MDA-MB-435S cells were cultured in supernatant from PBS-, EXO-PT- or EXO-TT (5 μg/ml for 6 h)-stimulated DCs with or without 30 μg/ml of anti-IL-6 or ISO mAbs for 24 h. Then, their invasive ability was measured using an in vitro invasive assay, and the cells on the bottom of the Transwell filter were imaged and quantified (Magnification: 200 ×). (D) The results of (C) were statistically analyzed (n = 5). (E, F) After adsorption onto latex beads, HSP72 and HSP105 on exosomes from sera of breast tumor patients or healthy controls were detected by flow cytometry. One representative result of 12 is shown (E). RFI was calculated and statistically analyzed (F). (B, D) The results are shown as the mean ± SEM of 3 independent experiments. (C) One representative of 3 independent experiments is shown. P values were generated by 2-tail student's t -test in A, F, and by one-way ANOVA, followed by a Tukey-Kramer multiple comparison test in B, D; *** p
    Figure Legend Snippet: Exosomes from tumor patients with increased membrane-associated HSP72 and HSP105 induced DCs to promote tumor cell invasion via IL-6 in vitro . (A) After adsorption onto latex beads, HSP72 and HSP105 on EXO-PT and EXO-TT were detected by flow cytometry. RFI was calculated by dividing mean fluorescence intensity of samples staining with detection Abs by that of sample staining with ISO Abs (n = 7). (B) Human DCs were induced from PBMCs and stimulated with 5 μg/ml of EXO-PT or EXO-TT from breast tumor tissues for 6 h. The IL-6 production was measured by ELISA (n = 3). (C) MDA-MB-435S cells were cultured in supernatant from PBS-, EXO-PT- or EXO-TT (5 μg/ml for 6 h)-stimulated DCs with or without 30 μg/ml of anti-IL-6 or ISO mAbs for 24 h. Then, their invasive ability was measured using an in vitro invasive assay, and the cells on the bottom of the Transwell filter were imaged and quantified (Magnification: 200 ×). (D) The results of (C) were statistically analyzed (n = 5). (E, F) After adsorption onto latex beads, HSP72 and HSP105 on exosomes from sera of breast tumor patients or healthy controls were detected by flow cytometry. One representative result of 12 is shown (E). RFI was calculated and statistically analyzed (F). (B, D) The results are shown as the mean ± SEM of 3 independent experiments. (C) One representative of 3 independent experiments is shown. P values were generated by 2-tail student's t -test in A, F, and by one-way ANOVA, followed by a Tukey-Kramer multiple comparison test in B, D; *** p

    Techniques Used: In Vitro, Adsorption, Flow Cytometry, Cytometry, Fluorescence, Staining, Enzyme-linked Immunosorbent Assay, Multiple Displacement Amplification, Cell Culture, Generated

    DCs with enhanced IL-6-secretion capacity were found in tumor-bearing mice and patients. (A, B) Mice were subcutaneously inoculated with 5 × 10 5 B16-F10 cells, and mice without tumor inoculation were used as controls. Then, DCs from splenocytes were isolated 1 week, 2 weeks or 3 weeks later and stimulated with PBS, 5 μg/ml B16-F10-EXO or 100 ng/ml of LPS for 6 h. IL-6 production by DCs was detected by ELISA (n = 3) (A). DCs from splenocytes were isolated 3 weeks later and cultured in vitro for 6 h. Culture supernatant was collected and B16-F10 cells were cultured in this supernatant for 24 h. Then, the invasive ability of B16-F10 cells was measured (n = 5). Control indicates B16-F10 cells without any treatment (B). SN-DC PBS or SN-DC tumor denotes supernatant from DCs of healthy control mice or tumor mice, respectively. (C) Mice were intravenously injected with 100 μl of PBS or 10 μg/100 μl of B16-F10-EXO. Then, DCs from splenocytes were isolated 12 or 24 h later and cultured in vitro for 6 h. IL-6 production by DCs was detected by ELISA (n = 3). (D) Human DCs were isolated from PBMCs from healthy volunteers (n = 27) or breast tumor patients (n = 18) and cultured in vitro for 6 h with or without 100 ng/ml LPS. IL-6 production by DCs was detected by ELISA. The results are shown as the mean ± SEM of 3 independent experiments or of all detected samples. P values were generated by a 2-tail student's t -test.
    Figure Legend Snippet: DCs with enhanced IL-6-secretion capacity were found in tumor-bearing mice and patients. (A, B) Mice were subcutaneously inoculated with 5 × 10 5 B16-F10 cells, and mice without tumor inoculation were used as controls. Then, DCs from splenocytes were isolated 1 week, 2 weeks or 3 weeks later and stimulated with PBS, 5 μg/ml B16-F10-EXO or 100 ng/ml of LPS for 6 h. IL-6 production by DCs was detected by ELISA (n = 3) (A). DCs from splenocytes were isolated 3 weeks later and cultured in vitro for 6 h. Culture supernatant was collected and B16-F10 cells were cultured in this supernatant for 24 h. Then, the invasive ability of B16-F10 cells was measured (n = 5). Control indicates B16-F10 cells without any treatment (B). SN-DC PBS or SN-DC tumor denotes supernatant from DCs of healthy control mice or tumor mice, respectively. (C) Mice were intravenously injected with 100 μl of PBS or 10 μg/100 μl of B16-F10-EXO. Then, DCs from splenocytes were isolated 12 or 24 h later and cultured in vitro for 6 h. IL-6 production by DCs was detected by ELISA (n = 3). (D) Human DCs were isolated from PBMCs from healthy volunteers (n = 27) or breast tumor patients (n = 18) and cultured in vitro for 6 h with or without 100 ng/ml LPS. IL-6 production by DCs was detected by ELISA. The results are shown as the mean ± SEM of 3 independent experiments or of all detected samples. P values were generated by a 2-tail student's t -test.

    Techniques Used: Mouse Assay, Isolation, Enzyme-linked Immunosorbent Assay, Cell Culture, In Vitro, Injection, Generated

    Related Articles

    other:

    Article Title: Human dendritic cell maturation and activation by a heat-killed recombinant yeast (Saccharomyces cerevisiae) vector encoding carcinoembryonic antigen
    Article Snippet: PBMCs were separated by lymphocyte separation medium gradient (MP Biomedicals, Aurora, OH) according to the manufacturer’s instructions.

    Article Title: Memory Stem T Cells in Autoimmune Disease: High Frequency of Circulating CD8+ Memory Stem Cells in Acquired Aplastic Anemia
    Article Snippet: PBMCs were separated from PB samples using Lymphocyte Separation Medium (MP Biomedicals LLC, CA) and cryopreserved in RPMI-1640 (Life Technologies, Gaithersburg, MD) supplemented with 20% heat-inactivated fetal bovine serum (Sigma-Aldrich, St Louis, MO) and 10% dimethyl sulfoxide, according to the standard protocol until use.

    Article Title: Comprehensive siRNA-based screening of human and mouse TLR pathways identifies species-specific preferences in signaling protein use
    Article Snippet: PBMCs were prepared by a 1:1 dilution of a human blood sample with Hank’s balanced salt solution (HBSS) and layering onto the surface of lymphocyte separation medium (MP Biomedicals, LLC, 50494) at a 2:1 ratio.

    Article Title: Caveolin-1 mediates cellular distribution of HER2 and affects trastuzumab binding and therapeutic efficacy
    Article Snippet: PBMCs, obtained from the Immune Monitoring Facility at MSKCC, were separated from the blood of normal volunteers using lymphocyte separation medium (MP Biomedicals).

    Article Title: Janus kinase-2 inhibition induces durable tolerance to alloantigen by human dendritic cell-stimulated T cells yet preserves immunity to recall antigen
    Article Snippet: PBMCs were stimulated with Con A (5 μg/mL; MP Biomedicals) for 48 hours to generate activated T-cell blasts, then harvested and washed with α-methylmannoside (10mM; Calbiochem) to remove the Con A.

    Purification:

    Article Title: Lentiviral hematopoietic stem cell gene therapy for X-linked severe combined immunodeficiency
    Article Snippet: B cell proliferation was determined by CFSE dilution, and immunoglobulin class switch was measured by intracellular expression of IgG and IgM using flow cytometry. .. PBMCs were purified from non-mobilized peripheral blood by Ficoll separation (Lymphocyte Separation Medium, MP Biomedicals). .. CD34+ cells were isolated from PBMCs using magnetic cell sorter magnetic beads (Miltenyi Biotec) according to the manufacturer’s protocol.

    Isolation:

    Article Title: The multi-functionality of N-809, a novel fusion protein encompassing anti-PD-L1 and the IL-15 superagonist fusion complex
    Article Snippet: .. PBMCs were isolated using the LSM Lymphocyte Separation Medium (MP Biomedicals, Santa Ana, CA), washed three times, and frozen at 5 × 10 cells/ml in 10% DMSO and 90% FBS at −80°C for 24 hours, then moved to liquid nitrogen for storage. .. Cell counts were performed on a Nexcelom Cellometer Auto 2000 (Nexcelom Bioscience, Lawrence, MA) with AO/PI staining.

    Article Title: Microglia Require CD4 T Cells to Complete the Fetal-to-Adult Transition
    Article Snippet: Lung tissue was treated similarly, while the small intestine was subjected to a pre-digest incubation in HBSS with 2% FCS and 10mM EDTA to remove the epithelial layer. .. PBMCs were isolated using LSM (MP Biomedicals) from healthy individuals. .. Non-specific binding was blocked using either 2.4G2 supernatant for mouse cells or normal mouse serum for human cells, and dead cells were labeled by fixable viability dye eFluor 780 (ThermoFisher).

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    • blood mononuclear cells pbmc  (Valiant)
    86
    Valiant blood mononuclear cells pbmc
    TV-antigen containing lymphocyte populations in cultured, TV-inoculated <t>PBMCs.</t> A B– One day-old cultures of TV-inoculated PBMCs show the trend (p > 0.05) for TV antigen expression by CD20+HLA-DR+ B cells. C D– Six days-old cultures show significant presence of TV antigens predominantly inside the CD20+HLA-DR+ cells (p
    Blood Mononuclear Cells Pbmc, supplied by Valiant, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/blood mononuclear cells pbmc/product/Valiant
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    blood mononuclear cells pbmc - by Bioz Stars, 2021-03
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    peripheral blood mononuclear cells pbmcs  (Valiant)
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    Valiant peripheral blood mononuclear cells pbmcs
    Isolation and molecular characterization of T cell subsets from <t>PNH</t> patients ( A ) Gating strategy for T cell subsets for cell sorting. <t>PBMCs</t> were stained with ViViD, anti-CD14-Pacific Blue, anti-CD19-Pacific Blue, anti-CD3-BV605, anti-CD4-V500, anti-CD8-APC-H7, anti-CD45RA-PE-Cy7, and anti-CD45RO-APC. Lymphocytes were gated based on their scatter characteristics or forward scatter height vs. forward scatter area. Live T cells were gated based on positive for CD3 and negative for ViViD, CD14, and CD19 to remove dead cells, monocytes, and B cells. CD4 + and CD8 + T cells were then gated based on the characteristic expression patterns of CD45RA and CD45RO. Four different T cell subsets [CD4 + naïve (CD45RA + CD45RO - ), CD4 + memory (CD45RA - CD45RO + ), CD8 + naïve (CD45RA + CD45RO - ), and CD8 + memory (CD45RA - CD45RO + ) T cells] were sorted for RNA-seq. ( B ) Transcriptional validation of the naïve and memory T cell identity by analyzing expression levels of CD4, CD8, CCR5, and EOMES. FPKM (fragments per kilobase of exon per million fragments mapped). * p
    Peripheral Blood Mononuclear Cells Pbmcs, supplied by Valiant, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/peripheral blood mononuclear cells pbmcs/product/Valiant
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    peripheral blood mononuclear cells pbmcs - by Bioz Stars, 2021-03
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    interleukin 2 il 2 treatment pbmcs  (Valiant)
    93
    Valiant interleukin 2 il 2 treatment pbmcs
    Cytotoxicity against a malignant pleural mesothelioma cell line mediated by cetuximab. (A) Cetuximab mediates cytotoxicity against the EGFR-expressing MSTO-211H mesothelioma cell line. Healthy human <t>PBMCs,</t> using 4 different E/T ratios, were tested for cytotoxicity in the presence or absence of cetuximab (2.5 μg/ml). The y-axis reveals cytotoxicity as determined by a 4-h 51 Cr release assays. (B) Concentration-dependent curve of cetuximab-dependent ADCC activity and NK activity against MSTO-211H cells by healthy human PBMCs. MSTO-211H cells were incubated with PBMCs at an E/T ratio of 20:1 along with or without indicated concentrations of cetuximab (0.0000025-1,000 μg/ml). Data are representative of 3 independent experiments. Points, mean of a triplicate experiment; bars, SD. (C) Correlation between EGFR expression levels of target malignant mesothelioma cell lines and cetuximab-mediated ADCC activity. The x-axis indicates the number of EGFR molecules expressed on the surface of the cancer cells. The y-axis represents the ADCC activity of cetuximab (0.25 mg/ml) as determined by a 4-h 51 Cr release assay. Healthy human PBMCs were incubated with or without <t>IL-2</t> (30 IU/ml) at an E/T ratio of 40:1 for 18 h and tested for cetuximab-mediated ADCC activity against various 51 Cr-labeled cell lines. Data are representative of 5 independent experiments. The malignant mesothelioma cell lines used are indicated. Points, mean of a triplicate experiment; bars, SD.
    Interleukin 2 Il 2 Treatment Pbmcs, supplied by Valiant, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TV-antigen containing lymphocyte populations in cultured, TV-inoculated PBMCs. A B– One day-old cultures of TV-inoculated PBMCs show the trend (p > 0.05) for TV antigen expression by CD20+HLA-DR+ B cells. C D– Six days-old cultures show significant presence of TV antigens predominantly inside the CD20+HLA-DR+ cells (p

    Journal: PLoS ONE

    Article Title: Experimental Inoculation of Juvenile Rhesus Macaques with Primate Enteric Caliciviruses

    doi: 10.1371/journal.pone.0037973

    Figure Lengend Snippet: TV-antigen containing lymphocyte populations in cultured, TV-inoculated PBMCs. A B– One day-old cultures of TV-inoculated PBMCs show the trend (p > 0.05) for TV antigen expression by CD20+HLA-DR+ B cells. C D– Six days-old cultures show significant presence of TV antigens predominantly inside the CD20+HLA-DR+ cells (p

    Article Snippet: TV infection of peripheral blood mononuclear cells (PBMC) with TV PBMCs were isolated from whole blood of 8 healthy, normal rhesus macaques by density gradient centrifugation using Lymphocyte Separation Medium (MP Biomedicals, LLC, Santa Ana, CA) and seeded in 24-well cell culture plates coated with collagen (BD Biosciences, San Diego, CA) at a concentration of 2×106 cells per well.

    Techniques: Cell Culture, Expressing

    Flow cytometry detection of TV antigens-containing cells in vitro . A– PBMCs isolated from healthy rhesus macaques were used for in vitro inoculation with TV. After being cultured in vitro for 6 days, cells were sorted out into CD3+ T cell and CD20+ B cell populations. Presence of TV antigens was detected in CD20+ B cells as shown by histograms (black peak). B– The CD20+ B cells were further subdivided into populations expressing the HLA-DR, CD11c or CD123 antigens. Presence of TV was revealed predominantly in CD20+HLA-DR+ cells while some of the other lymphocyte populations including the CD20+CD11c+ and CD20+CD123+ B cells also contained TV antigens.

    Journal: PLoS ONE

    Article Title: Experimental Inoculation of Juvenile Rhesus Macaques with Primate Enteric Caliciviruses

    doi: 10.1371/journal.pone.0037973

    Figure Lengend Snippet: Flow cytometry detection of TV antigens-containing cells in vitro . A– PBMCs isolated from healthy rhesus macaques were used for in vitro inoculation with TV. After being cultured in vitro for 6 days, cells were sorted out into CD3+ T cell and CD20+ B cell populations. Presence of TV antigens was detected in CD20+ B cells as shown by histograms (black peak). B– The CD20+ B cells were further subdivided into populations expressing the HLA-DR, CD11c or CD123 antigens. Presence of TV was revealed predominantly in CD20+HLA-DR+ cells while some of the other lymphocyte populations including the CD20+CD11c+ and CD20+CD123+ B cells also contained TV antigens.

    Article Snippet: TV infection of peripheral blood mononuclear cells (PBMC) with TV PBMCs were isolated from whole blood of 8 healthy, normal rhesus macaques by density gradient centrifugation using Lymphocyte Separation Medium (MP Biomedicals, LLC, Santa Ana, CA) and seeded in 24-well cell culture plates coated with collagen (BD Biosciences, San Diego, CA) at a concentration of 2×106 cells per well.

    Techniques: Flow Cytometry, Cytometry, In Vitro, Isolation, Cell Culture, Expressing

    Isolation and molecular characterization of T cell subsets from PNH patients ( A ) Gating strategy for T cell subsets for cell sorting. PBMCs were stained with ViViD, anti-CD14-Pacific Blue, anti-CD19-Pacific Blue, anti-CD3-BV605, anti-CD4-V500, anti-CD8-APC-H7, anti-CD45RA-PE-Cy7, and anti-CD45RO-APC. Lymphocytes were gated based on their scatter characteristics or forward scatter height vs. forward scatter area. Live T cells were gated based on positive for CD3 and negative for ViViD, CD14, and CD19 to remove dead cells, monocytes, and B cells. CD4 + and CD8 + T cells were then gated based on the characteristic expression patterns of CD45RA and CD45RO. Four different T cell subsets [CD4 + naïve (CD45RA + CD45RO - ), CD4 + memory (CD45RA - CD45RO + ), CD8 + naïve (CD45RA + CD45RO - ), and CD8 + memory (CD45RA - CD45RO + ) T cells] were sorted for RNA-seq. ( B ) Transcriptional validation of the naïve and memory T cell identity by analyzing expression levels of CD4, CD8, CCR5, and EOMES. FPKM (fragments per kilobase of exon per million fragments mapped). * p

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: T Cell Transcriptomes from Paroxysmal Nocturnal Hemoglobinuria Patients Reveal Novel Signaling Pathways

    doi: 10.4049/jimmunol.1601299

    Figure Lengend Snippet: Isolation and molecular characterization of T cell subsets from PNH patients ( A ) Gating strategy for T cell subsets for cell sorting. PBMCs were stained with ViViD, anti-CD14-Pacific Blue, anti-CD19-Pacific Blue, anti-CD3-BV605, anti-CD4-V500, anti-CD8-APC-H7, anti-CD45RA-PE-Cy7, and anti-CD45RO-APC. Lymphocytes were gated based on their scatter characteristics or forward scatter height vs. forward scatter area. Live T cells were gated based on positive for CD3 and negative for ViViD, CD14, and CD19 to remove dead cells, monocytes, and B cells. CD4 + and CD8 + T cells were then gated based on the characteristic expression patterns of CD45RA and CD45RO. Four different T cell subsets [CD4 + naïve (CD45RA + CD45RO - ), CD4 + memory (CD45RA - CD45RO + ), CD8 + naïve (CD45RA + CD45RO - ), and CD8 + memory (CD45RA - CD45RO + ) T cells] were sorted for RNA-seq. ( B ) Transcriptional validation of the naïve and memory T cell identity by analyzing expression levels of CD4, CD8, CCR5, and EOMES. FPKM (fragments per kilobase of exon per million fragments mapped). * p

    Article Snippet: For cell sorting, peripheral blood mononuclear cells (PBMCs) from PNH patients and healthy controls were separated from PB samples using Lymphocyte Separation Medium (MP Biomedicals, Santa Ana, CA).

    Techniques: Isolation, FACS, Staining, Expressing, RNA Sequencing Assay

    Exosomes from tumor patients with increased membrane-associated HSP72 and HSP105 induced DCs to promote tumor cell invasion via IL-6 in vitro . (A) After adsorption onto latex beads, HSP72 and HSP105 on EXO-PT and EXO-TT were detected by flow cytometry. RFI was calculated by dividing mean fluorescence intensity of samples staining with detection Abs by that of sample staining with ISO Abs (n = 7). (B) Human DCs were induced from PBMCs and stimulated with 5 μg/ml of EXO-PT or EXO-TT from breast tumor tissues for 6 h. The IL-6 production was measured by ELISA (n = 3). (C) MDA-MB-435S cells were cultured in supernatant from PBS-, EXO-PT- or EXO-TT (5 μg/ml for 6 h)-stimulated DCs with or without 30 μg/ml of anti-IL-6 or ISO mAbs for 24 h. Then, their invasive ability was measured using an in vitro invasive assay, and the cells on the bottom of the Transwell filter were imaged and quantified (Magnification: 200 ×). (D) The results of (C) were statistically analyzed (n = 5). (E, F) After adsorption onto latex beads, HSP72 and HSP105 on exosomes from sera of breast tumor patients or healthy controls were detected by flow cytometry. One representative result of 12 is shown (E). RFI was calculated and statistically analyzed (F). (B, D) The results are shown as the mean ± SEM of 3 independent experiments. (C) One representative of 3 independent experiments is shown. P values were generated by 2-tail student's t -test in A, F, and by one-way ANOVA, followed by a Tukey-Kramer multiple comparison test in B, D; *** p

    Journal: Oncoimmunology

    Article Title: Tumor-derived exosomes educate dendritic cells to promote tumor metastasis via HSP72/HSP105-TLR2/TLR4 pathway

    doi: 10.1080/2162402X.2017.1362527

    Figure Lengend Snippet: Exosomes from tumor patients with increased membrane-associated HSP72 and HSP105 induced DCs to promote tumor cell invasion via IL-6 in vitro . (A) After adsorption onto latex beads, HSP72 and HSP105 on EXO-PT and EXO-TT were detected by flow cytometry. RFI was calculated by dividing mean fluorescence intensity of samples staining with detection Abs by that of sample staining with ISO Abs (n = 7). (B) Human DCs were induced from PBMCs and stimulated with 5 μg/ml of EXO-PT or EXO-TT from breast tumor tissues for 6 h. The IL-6 production was measured by ELISA (n = 3). (C) MDA-MB-435S cells were cultured in supernatant from PBS-, EXO-PT- or EXO-TT (5 μg/ml for 6 h)-stimulated DCs with or without 30 μg/ml of anti-IL-6 or ISO mAbs for 24 h. Then, their invasive ability was measured using an in vitro invasive assay, and the cells on the bottom of the Transwell filter were imaged and quantified (Magnification: 200 ×). (D) The results of (C) were statistically analyzed (n = 5). (E, F) After adsorption onto latex beads, HSP72 and HSP105 on exosomes from sera of breast tumor patients or healthy controls were detected by flow cytometry. One representative result of 12 is shown (E). RFI was calculated and statistically analyzed (F). (B, D) The results are shown as the mean ± SEM of 3 independent experiments. (C) One representative of 3 independent experiments is shown. P values were generated by 2-tail student's t -test in A, F, and by one-way ANOVA, followed by a Tukey-Kramer multiple comparison test in B, D; *** p

    Article Snippet: To generate human DCs, peripheral blood mononuclear cells (PBMCs) from healthy volunteers were isolated by density centrifugation of heparinized blood on LSM® (MP Biomedicals), resuspended in culture medium and allowed to adhere in 6-well plates.

    Techniques: In Vitro, Adsorption, Flow Cytometry, Cytometry, Fluorescence, Staining, Enzyme-linked Immunosorbent Assay, Multiple Displacement Amplification, Cell Culture, Generated

    DCs with enhanced IL-6-secretion capacity were found in tumor-bearing mice and patients. (A, B) Mice were subcutaneously inoculated with 5 × 10 5 B16-F10 cells, and mice without tumor inoculation were used as controls. Then, DCs from splenocytes were isolated 1 week, 2 weeks or 3 weeks later and stimulated with PBS, 5 μg/ml B16-F10-EXO or 100 ng/ml of LPS for 6 h. IL-6 production by DCs was detected by ELISA (n = 3) (A). DCs from splenocytes were isolated 3 weeks later and cultured in vitro for 6 h. Culture supernatant was collected and B16-F10 cells were cultured in this supernatant for 24 h. Then, the invasive ability of B16-F10 cells was measured (n = 5). Control indicates B16-F10 cells without any treatment (B). SN-DC PBS or SN-DC tumor denotes supernatant from DCs of healthy control mice or tumor mice, respectively. (C) Mice were intravenously injected with 100 μl of PBS or 10 μg/100 μl of B16-F10-EXO. Then, DCs from splenocytes were isolated 12 or 24 h later and cultured in vitro for 6 h. IL-6 production by DCs was detected by ELISA (n = 3). (D) Human DCs were isolated from PBMCs from healthy volunteers (n = 27) or breast tumor patients (n = 18) and cultured in vitro for 6 h with or without 100 ng/ml LPS. IL-6 production by DCs was detected by ELISA. The results are shown as the mean ± SEM of 3 independent experiments or of all detected samples. P values were generated by a 2-tail student's t -test.

    Journal: Oncoimmunology

    Article Title: Tumor-derived exosomes educate dendritic cells to promote tumor metastasis via HSP72/HSP105-TLR2/TLR4 pathway

    doi: 10.1080/2162402X.2017.1362527

    Figure Lengend Snippet: DCs with enhanced IL-6-secretion capacity were found in tumor-bearing mice and patients. (A, B) Mice were subcutaneously inoculated with 5 × 10 5 B16-F10 cells, and mice without tumor inoculation were used as controls. Then, DCs from splenocytes were isolated 1 week, 2 weeks or 3 weeks later and stimulated with PBS, 5 μg/ml B16-F10-EXO or 100 ng/ml of LPS for 6 h. IL-6 production by DCs was detected by ELISA (n = 3) (A). DCs from splenocytes were isolated 3 weeks later and cultured in vitro for 6 h. Culture supernatant was collected and B16-F10 cells were cultured in this supernatant for 24 h. Then, the invasive ability of B16-F10 cells was measured (n = 5). Control indicates B16-F10 cells without any treatment (B). SN-DC PBS or SN-DC tumor denotes supernatant from DCs of healthy control mice or tumor mice, respectively. (C) Mice were intravenously injected with 100 μl of PBS or 10 μg/100 μl of B16-F10-EXO. Then, DCs from splenocytes were isolated 12 or 24 h later and cultured in vitro for 6 h. IL-6 production by DCs was detected by ELISA (n = 3). (D) Human DCs were isolated from PBMCs from healthy volunteers (n = 27) or breast tumor patients (n = 18) and cultured in vitro for 6 h with or without 100 ng/ml LPS. IL-6 production by DCs was detected by ELISA. The results are shown as the mean ± SEM of 3 independent experiments or of all detected samples. P values were generated by a 2-tail student's t -test.

    Article Snippet: To generate human DCs, peripheral blood mononuclear cells (PBMCs) from healthy volunteers were isolated by density centrifugation of heparinized blood on LSM® (MP Biomedicals), resuspended in culture medium and allowed to adhere in 6-well plates.

    Techniques: Mouse Assay, Isolation, Enzyme-linked Immunosorbent Assay, Cell Culture, In Vitro, Injection, Generated

    Cytotoxicity against a malignant pleural mesothelioma cell line mediated by cetuximab. (A) Cetuximab mediates cytotoxicity against the EGFR-expressing MSTO-211H mesothelioma cell line. Healthy human PBMCs, using 4 different E/T ratios, were tested for cytotoxicity in the presence or absence of cetuximab (2.5 μg/ml). The y-axis reveals cytotoxicity as determined by a 4-h 51 Cr release assays. (B) Concentration-dependent curve of cetuximab-dependent ADCC activity and NK activity against MSTO-211H cells by healthy human PBMCs. MSTO-211H cells were incubated with PBMCs at an E/T ratio of 20:1 along with or without indicated concentrations of cetuximab (0.0000025-1,000 μg/ml). Data are representative of 3 independent experiments. Points, mean of a triplicate experiment; bars, SD. (C) Correlation between EGFR expression levels of target malignant mesothelioma cell lines and cetuximab-mediated ADCC activity. The x-axis indicates the number of EGFR molecules expressed on the surface of the cancer cells. The y-axis represents the ADCC activity of cetuximab (0.25 mg/ml) as determined by a 4-h 51 Cr release assay. Healthy human PBMCs were incubated with or without IL-2 (30 IU/ml) at an E/T ratio of 40:1 for 18 h and tested for cetuximab-mediated ADCC activity against various 51 Cr-labeled cell lines. Data are representative of 5 independent experiments. The malignant mesothelioma cell lines used are indicated. Points, mean of a triplicate experiment; bars, SD.

    Journal: International Journal of Oncology

    Article Title: Therapeutic antitumor efficacy of anti-epidermal growth factor receptor antibody, cetuximab, against malignant pleural mesothelioma

    doi: 10.3892/ijo.2012.1607

    Figure Lengend Snippet: Cytotoxicity against a malignant pleural mesothelioma cell line mediated by cetuximab. (A) Cetuximab mediates cytotoxicity against the EGFR-expressing MSTO-211H mesothelioma cell line. Healthy human PBMCs, using 4 different E/T ratios, were tested for cytotoxicity in the presence or absence of cetuximab (2.5 μg/ml). The y-axis reveals cytotoxicity as determined by a 4-h 51 Cr release assays. (B) Concentration-dependent curve of cetuximab-dependent ADCC activity and NK activity against MSTO-211H cells by healthy human PBMCs. MSTO-211H cells were incubated with PBMCs at an E/T ratio of 20:1 along with or without indicated concentrations of cetuximab (0.0000025-1,000 μg/ml). Data are representative of 3 independent experiments. Points, mean of a triplicate experiment; bars, SD. (C) Correlation between EGFR expression levels of target malignant mesothelioma cell lines and cetuximab-mediated ADCC activity. The x-axis indicates the number of EGFR molecules expressed on the surface of the cancer cells. The y-axis represents the ADCC activity of cetuximab (0.25 mg/ml) as determined by a 4-h 51 Cr release assay. Healthy human PBMCs were incubated with or without IL-2 (30 IU/ml) at an E/T ratio of 40:1 for 18 h and tested for cetuximab-mediated ADCC activity against various 51 Cr-labeled cell lines. Data are representative of 5 independent experiments. The malignant mesothelioma cell lines used are indicated. Points, mean of a triplicate experiment; bars, SD.

    Article Snippet: Isolation of peripheral blood mononuclear cells (PBMCs) and interleukin-2 (IL-2) treatment PBMCs were isolated from heparinized peripheral blood by lymphocyte-separation-medium (MP Biomedicals, Irvine, CA, USA) density gradient centrifugation.

    Techniques: Expressing, Concentration Assay, Activity Assay, Incubation, Release Assay, Labeling

    Effect of cetuximab alone and in combination with IL-2 on SCID mice bearing MSTO-211H cells. Mice were treated with cetuximab (0.05 mg/mouse i.t. on day 7) or in combination with IL-2 (30 IU/ml i.t. on day 7). (A) Representative intrathoracic pictures of SCID mice bearing MSTO-211H cells on day 21. The MSTO-211H cells produced small nodular tumors intrathoracic cavity. (B) Tumor area of the mice treated with cetuximab or in combination with IL-2 on day 21. Columns, mean pixels from 5 independent animals; bars, SD. * P

    Journal: International Journal of Oncology

    Article Title: Therapeutic antitumor efficacy of anti-epidermal growth factor receptor antibody, cetuximab, against malignant pleural mesothelioma

    doi: 10.3892/ijo.2012.1607

    Figure Lengend Snippet: Effect of cetuximab alone and in combination with IL-2 on SCID mice bearing MSTO-211H cells. Mice were treated with cetuximab (0.05 mg/mouse i.t. on day 7) or in combination with IL-2 (30 IU/ml i.t. on day 7). (A) Representative intrathoracic pictures of SCID mice bearing MSTO-211H cells on day 21. The MSTO-211H cells produced small nodular tumors intrathoracic cavity. (B) Tumor area of the mice treated with cetuximab or in combination with IL-2 on day 21. Columns, mean pixels from 5 independent animals; bars, SD. * P

    Article Snippet: Isolation of peripheral blood mononuclear cells (PBMCs) and interleukin-2 (IL-2) treatment PBMCs were isolated from heparinized peripheral blood by lymphocyte-separation-medium (MP Biomedicals, Irvine, CA, USA) density gradient centrifugation.

    Techniques: Mouse Assay, Produced