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cd16  (Miltenyi Biotec)


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    Miltenyi Biotec cd16
    DNA methylation and isoform-specific expression within the promoter regions of <t>FCGR3A</t> and FCGR3B. A) Representation of the <t>FCGR3</t> locus spanning approximately 100 kb of chromosome 1q23.3 displaying transcripts annotated in the RefSeq database (adapted from the UCSC Genome Browser). Below, promoter regions of FCGR3A and FCGR3B are enlarged displaying the various transcript variants annotated in the RefSeq database that differ in the 5′ region. MassARRAY amplicons (green bars) used are shown along with the positions of CpG dinucleotides (green markers). TSS, transcriptional start site. B) The MassARRAY EpiTYPER assay was used to interrogate the DNA methylation levels of CpGs across the promoter region of FCGR3A and FCGR3B in a gene-specific manner in sorted NK CD16a+ and CD16a- fractions. Methylation plots are aligned to enlargements of gene promoters (in B) according to the hg19 genome assembly. C, D) DNA methylation levels in NK cell lines and neutrophils, respectively. E) The expression ratio of FCGR3A to FCGR3B in CD16a+ NK cells and neutrophils as determined by the MassARRAY iPLEX assay. F) Quantitative RT-PCR analysis of 5′ transcript variants using primers specific for variant 1 or variants 3 and 2 indicating promoter usage in NK cells, neutrophils, and NK cell lines YT and NKL (both primer pairs do not distinguish FCGR3A vs. FCGR3B). D1-4 represents donor numbers 1–4. Expression values expressed relative to the average of four housekeeping (HK) genes.
    Cd16, supplied by Miltenyi Biotec, used in various techniques. Bioz Stars score: 95/100, based on 108 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/massarray+quantitative+methylation+analysis+platform/CD16+Antibody%2C+anti-human/pmc05881939-64-26-28
    Average 95 stars, based on 108 article reviews
    cd16 - by Bioz Stars, 2026-10
    95/100 stars

    Images

    1) Product Images from "Epigenetic and post-transcriptional regulation of CD16 expression during human natural killer cell development 1 "

    Article Title: Epigenetic and post-transcriptional regulation of CD16 expression during human natural killer cell development 1

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

    doi: 10.4049/jimmunol.1701128

    DNA methylation and isoform-specific expression within the promoter regions of FCGR3A and FCGR3B. A) Representation of the FCGR3 locus spanning approximately 100 kb of chromosome 1q23.3 displaying transcripts annotated in the RefSeq database (adapted from the UCSC Genome Browser). Below, promoter regions of FCGR3A and FCGR3B are enlarged displaying the various transcript variants annotated in the RefSeq database that differ in the 5′ region. MassARRAY amplicons (green bars) used are shown along with the positions of CpG dinucleotides (green markers). TSS, transcriptional start site. B) The MassARRAY EpiTYPER assay was used to interrogate the DNA methylation levels of CpGs across the promoter region of FCGR3A and FCGR3B in a gene-specific manner in sorted NK CD16a+ and CD16a- fractions. Methylation plots are aligned to enlargements of gene promoters (in B) according to the hg19 genome assembly. C, D) DNA methylation levels in NK cell lines and neutrophils, respectively. E) The expression ratio of FCGR3A to FCGR3B in CD16a+ NK cells and neutrophils as determined by the MassARRAY iPLEX assay. F) Quantitative RT-PCR analysis of 5′ transcript variants using primers specific for variant 1 or variants 3 and 2 indicating promoter usage in NK cells, neutrophils, and NK cell lines YT and NKL (both primer pairs do not distinguish FCGR3A vs. FCGR3B). D1-4 represents donor numbers 1–4. Expression values expressed relative to the average of four housekeeping (HK) genes.
    Figure Legend Snippet: DNA methylation and isoform-specific expression within the promoter regions of FCGR3A and FCGR3B. A) Representation of the FCGR3 locus spanning approximately 100 kb of chromosome 1q23.3 displaying transcripts annotated in the RefSeq database (adapted from the UCSC Genome Browser). Below, promoter regions of FCGR3A and FCGR3B are enlarged displaying the various transcript variants annotated in the RefSeq database that differ in the 5′ region. MassARRAY amplicons (green bars) used are shown along with the positions of CpG dinucleotides (green markers). TSS, transcriptional start site. B) The MassARRAY EpiTYPER assay was used to interrogate the DNA methylation levels of CpGs across the promoter region of FCGR3A and FCGR3B in a gene-specific manner in sorted NK CD16a+ and CD16a- fractions. Methylation plots are aligned to enlargements of gene promoters (in B) according to the hg19 genome assembly. C, D) DNA methylation levels in NK cell lines and neutrophils, respectively. E) The expression ratio of FCGR3A to FCGR3B in CD16a+ NK cells and neutrophils as determined by the MassARRAY iPLEX assay. F) Quantitative RT-PCR analysis of 5′ transcript variants using primers specific for variant 1 or variants 3 and 2 indicating promoter usage in NK cells, neutrophils, and NK cell lines YT and NKL (both primer pairs do not distinguish FCGR3A vs. FCGR3B). D1-4 represents donor numbers 1–4. Expression values expressed relative to the average of four housekeeping (HK) genes.

    Techniques Used: DNA Methylation Assay, Expressing, MassARRAY EpiTYPER assay, Methylation, Quantitative RT-PCR, Variant Assay

    Analysis of DNA methylation- and lineage-specific activity of FCGR3 promoter sequences. A) Illustration of FCGR3A and FCGR3B sequences cloned into luciferase constructs (black bars). B) Four separate promoter sequence fragments were cloned from either FCGR3A (left) or FCGR3B (right) and transfected in various cell lines. Luciferase assays showing sequence- and gene-specific activity (relative to empty-vector control). Pmed1-A and Pmed1-B were additionally methylated in vitro prior to transfection. Error bars represent SEM of n=3 individual experiments; ND, not done. C) Sequence alignment of Pmed1-A and Pmed1-B with numbered CpGs. All sequence variants are enlarged below; an 8 bp repeat occurring in the vicinity of CpG#6 is highlighted in blue, asterisks below the sequence indicate homology. D) Luciferase activity following site-directed mutagenesis of CpG#s 1&2 of Pmed1-A compared to unaltered FCGR3A and FCGR3B sequences in YT and K562 cells.
    Figure Legend Snippet: Analysis of DNA methylation- and lineage-specific activity of FCGR3 promoter sequences. A) Illustration of FCGR3A and FCGR3B sequences cloned into luciferase constructs (black bars). B) Four separate promoter sequence fragments were cloned from either FCGR3A (left) or FCGR3B (right) and transfected in various cell lines. Luciferase assays showing sequence- and gene-specific activity (relative to empty-vector control). Pmed1-A and Pmed1-B were additionally methylated in vitro prior to transfection. Error bars represent SEM of n=3 individual experiments; ND, not done. C) Sequence alignment of Pmed1-A and Pmed1-B with numbered CpGs. All sequence variants are enlarged below; an 8 bp repeat occurring in the vicinity of CpG#6 is highlighted in blue, asterisks below the sequence indicate homology. D) Luciferase activity following site-directed mutagenesis of CpG#s 1&2 of Pmed1-A compared to unaltered FCGR3A and FCGR3B sequences in YT and K562 cells.

    Techniques Used: DNA Methylation Assay, Activity Assay, Clone Assay, Luciferase, Construct, Sequencing, Transfection, Plasmid Preparation, Control, Methylation, In Vitro, Mutagenesis

    Identification of miR-218 as a potential regulator of FCGR3A. (A) Expression ratio of predicted miRNAs that were present in the miRNA expression array comparing CD16a+ and CD16a- NK cells freshly isolated from adult peripheral blood. A ratio <1 indicates low expression in CD16a+ NK cells while a ratio >1 indicates high expression in CD16a+ NK cells. (B) Predicted miRNA regulators of FCGR3A have putative binding sites in the FCGR3A 3′ UTR. (C) Validation of expression of predicted miRNA regulators of FCGR3A by qPCR (n=3). (D) Luciferase expression as a ratio of firefly/renilla for each expression vector (n=2). Data are presented as mean±SD, * indicates p<0.05.
    Figure Legend Snippet: Identification of miR-218 as a potential regulator of FCGR3A. (A) Expression ratio of predicted miRNAs that were present in the miRNA expression array comparing CD16a+ and CD16a- NK cells freshly isolated from adult peripheral blood. A ratio <1 indicates low expression in CD16a+ NK cells while a ratio >1 indicates high expression in CD16a+ NK cells. (B) Predicted miRNA regulators of FCGR3A have putative binding sites in the FCGR3A 3′ UTR. (C) Validation of expression of predicted miRNA regulators of FCGR3A by qPCR (n=3). (D) Luciferase expression as a ratio of firefly/renilla for each expression vector (n=2). Data are presented as mean±SD, * indicates p<0.05.

    Techniques Used: Expressing, Isolation, Binding Assay, Biomarker Discovery, Luciferase, Plasmid Preparation

    MiR-218 negatively regulates CD16a in primary human NK cells. Primary human NK cells were enriched by magnetic selection to >70% purity and infected with lentivirus containing either miR-218 or empty vector. 48hr after infection, NK cells were sorted as live/CD3-/CD56+/GFP+ lymphocytes. (A) Representative histogram plot (of one of six donors) of CD16a expression in live/CD3-/CD56+/GFP+ primary human NK cells infected with miR-218 or empty vector virus. (B) CD16a expression in primary human NK cells infected with miR-218 or empty vector virus (* indicates p=0.05). (C) Validation of miR-218 over-expression by real-time PCR (** indicates p<0.01). (D) FCGR3A mRNA expression assessed by real-time RT-PCR in sorted NK cells infected with either miR-218 or empty vector (*** indicates p<0.005). (B–D) Data are presented as mean±SD, n=6.
    Figure Legend Snippet: MiR-218 negatively regulates CD16a in primary human NK cells. Primary human NK cells were enriched by magnetic selection to >70% purity and infected with lentivirus containing either miR-218 or empty vector. 48hr after infection, NK cells were sorted as live/CD3-/CD56+/GFP+ lymphocytes. (A) Representative histogram plot (of one of six donors) of CD16a expression in live/CD3-/CD56+/GFP+ primary human NK cells infected with miR-218 or empty vector virus. (B) CD16a expression in primary human NK cells infected with miR-218 or empty vector virus (* indicates p=0.05). (C) Validation of miR-218 over-expression by real-time PCR (** indicates p<0.01). (D) FCGR3A mRNA expression assessed by real-time RT-PCR in sorted NK cells infected with either miR-218 or empty vector (*** indicates p<0.005). (B–D) Data are presented as mean±SD, n=6.

    Techniques Used: Selection, Infection, Plasmid Preparation, Expressing, Virus, Biomarker Discovery, Over Expression, Real-time Polymerase Chain Reaction, Quantitative RT-PCR

    Related Articles

    Magnetic Cell Separation:

    Article Title: Platelet extracellular vesicles drive monocyte differentiation into inflammatory foam cells in peripheral arterial disease.
    Article Snippet: .. Collected cells were resuspended in 400 ml MACS buffer and incubated with anti-CD16 human microbeads at 4◦C for 20 min. CD16+ monocytes were then positively selected using as LS column (Miltenyi; see above) and eluted using 5 ml MACS buffer, followed by cell counting. .. Monocytes were resuspended in pre-warmed 0.15 % (v/v) BSA (Gibco) in RPMI (Gibco) at a concentration of 7.0 × 105/ml for culture in a 24 well tissue culture plate (Falcon) or 7.5 × 105/ml for culture in 8 well μ-slide chambers (Ibidi).

    Article Title: Platelet extracellular vesicles drive monocyte differentiation into inflammatory foam cells in peripheral arterial disease.
    Article Snippet: .. Isolation of CD14+ CD16- monocytes used labelling of PBMCs with anti-CD16 human microbeads (Miltenyi) in MACS buffer at a concentration of 50 ml per 5 × 107 cells. .. After 20 min at 4◦C, CD16+ cells were depleted by passing them through a magnetic LD column (Miltenyi) according to manufacturer’s instructions.

    Incubation:

    Article Title: Platelet extracellular vesicles drive monocyte differentiation into inflammatory foam cells in peripheral arterial disease.
    Article Snippet: .. Collected cells were resuspended in 400 ml MACS buffer and incubated with anti-CD16 human microbeads at 4◦C for 20 min. CD16+ monocytes were then positively selected using as LS column (Miltenyi; see above) and eluted using 5 ml MACS buffer, followed by cell counting. .. Monocytes were resuspended in pre-warmed 0.15 % (v/v) BSA (Gibco) in RPMI (Gibco) at a concentration of 7.0 × 105/ml for culture in a 24 well tissue culture plate (Falcon) or 7.5 × 105/ml for culture in 8 well μ-slide chambers (Ibidi).

    Article Title: SFV Replicon Vector Harbouring Porcine Epidemic Diarrhoea Virus Immunogens Delivered by Attenuated Salmonella Typhimurium Induces PEDV Neutralising Antibodies and Lactogenic Immunogenicity in BALB/c Mice
    Article Snippet: For stimulation, 2 × 10 5 viable cells/well were seeded in round-bottom 96-well plates (SPL life sciences, Republic of Korea) in complete RPMI 1640 medium and stimulated with recombinant PEDV S1-NTD (1 μg/well) and PEDV-N (1 μg/well) proteins for 72 h at 37 °C, 5% CO 2 . .. Fc Block solution was prepared using anti-CD16 (Miltenyi Biotec, Bergisch Gladbach, Germany) in FACS buffer, and cells will be incubated for 15 min at 4 °C. .. The cells were then stained with a cocktail containing anti-mouse CD3a-PE (Miltenyi Biotec, Germany), CD8-FITC (Miltenyi Biotec, Germany), and CD4-perCP-vio700 (Miltenyi Biotec, Germany) and incubated for 30 min at 4 °C in the dark.

    Article Title: SFV Replicon Vector Harbouring Porcine Epidemic Diarrhoea Virus Immunogens Delivered by Attenuated Salmonella Typhimurium Induces PEDV Neutralising Antibodies and Lactogenic Immunogenicity in BALB/c Mice.
    Article Snippet: For stimulation, 2 × 105 viable cells/well were seeded in round-bottom 96-well plates (SPL life sciences, Republic of Korea) in complete RPMI 1640 medium and stimulated with recombinant PEDV S1-NTD (1 μg/well) and PEDV-N (1 μg/well) proteins for 72 h at 37 ◦C, 5% CO2. .. Fc Block solution was prepared using anti-CD16 (Miltenyi Biotec, Bergisch Gladbach, Germany) in FACS buffer, and cells will be incubated for 15 min at 4 ◦C. .. The cells were then stained with a cocktail containing anti-mouse CD3a-PE (Miltenyi Biotec, Germany), CD8-FITC (Miltenyi Biotec, Germany), and CD4-perCP-vio700 (Miltenyi Biotec, Germany) and incubated for 30 min at 4 ◦C in the dark.

    Cell Counting:

    Article Title: Platelet extracellular vesicles drive monocyte differentiation into inflammatory foam cells in peripheral arterial disease.
    Article Snippet: .. Collected cells were resuspended in 400 ml MACS buffer and incubated with anti-CD16 human microbeads at 4◦C for 20 min. CD16+ monocytes were then positively selected using as LS column (Miltenyi; see above) and eluted using 5 ml MACS buffer, followed by cell counting. .. Monocytes were resuspended in pre-warmed 0.15 % (v/v) BSA (Gibco) in RPMI (Gibco) at a concentration of 7.0 × 105/ml for culture in a 24 well tissue culture plate (Falcon) or 7.5 × 105/ml for culture in 8 well μ-slide chambers (Ibidi).

    Isolation:

    Article Title: Single domain antibodies and their use in cancer therapies
    Article Snippet: T Cell Isolation and Transduction Peripheral blood mononuclear cells (PBMCs) were recovered using the density gradient LymphoprepTM (StemCells). .. CD8+ T cells were isolated by negative selection using a cocktail containing antibodies against CD4, CD15, CD16, CD19, CD34, CD36, CD56, CD123, TCRγ/δ, and CD235a (Glycophorin A), according to the instruction of CD8+ T cell human isolation kit (Miltenyi). .. Isolated CD8+ T cells were then cultured in X-VIVO® medium (Lonza) supplemented with 50 μM of β-mercaptoethanol (Merck Millipore) and 5% Human Serum (Merck Millipore) and activated using Human T-activator CD3/CD28 Dynabeads® (Gibco) following the manufacture instructions.

    Article Title: Platelet extracellular vesicles drive monocyte differentiation into inflammatory foam cells in peripheral arterial disease.
    Article Snippet: .. Isolation of CD14+ CD16- monocytes used labelling of PBMCs with anti-CD16 human microbeads (Miltenyi) in MACS buffer at a concentration of 50 ml per 5 × 107 cells. .. After 20 min at 4◦C, CD16+ cells were depleted by passing them through a magnetic LD column (Miltenyi) according to manufacturer’s instructions.

    Selection:

    Article Title: Single domain antibodies and their use in cancer therapies
    Article Snippet: T Cell Isolation and Transduction Peripheral blood mononuclear cells (PBMCs) were recovered using the density gradient LymphoprepTM (StemCells). .. CD8+ T cells were isolated by negative selection using a cocktail containing antibodies against CD4, CD15, CD16, CD19, CD34, CD36, CD56, CD123, TCRγ/δ, and CD235a (Glycophorin A), according to the instruction of CD8+ T cell human isolation kit (Miltenyi). .. Isolated CD8+ T cells were then cultured in X-VIVO® medium (Lonza) supplemented with 50 μM of β-mercaptoethanol (Merck Millipore) and 5% Human Serum (Merck Millipore) and activated using Human T-activator CD3/CD28 Dynabeads® (Gibco) following the manufacture instructions.

    other:

    Article Title: DNAM-1 immunoreceptor integrates innate and adaptive immune programs to drive intestinal inflammation
    Article Snippet: Anti-TNF-α mAb (XT3.11) was purchased from Selleck (Houston, USA). mAbs against mouse CD49b (DX5), CD11b (M1/70), CD86 (GL1), CD155 (3F1), rat IgG2a, rat IgG1, hamster IgG1, hamster IgG2 (KKLH), rat IgG2a (RTK2758), Ly-6G (1A8), CD40 (3/23), CD112 (829038), Gr-1 (RB6-8C5), CD90.2 (53-2.1), CD11c (HL3), CD11b (M1/70), Ly-6C (AL-21), NKp46 (29A1.4), RORγt (Q31-378), IFN-γ (XMG1.2), IL-17A (TC11-18H10), IL-12(p40/p70) (C11.5), human CD19 (HIB19), CD138 (MI15), CD117 (104D2) and Fluorochrome-conjugated streptavidin were purchased from BD Biosciences (Franklin Lakes, NJ). mAb against T1/ST2 (DJ8) was purchased from MD Bioproducts (St. Paul, MN). mAb against mouse IL-22 (1H8PWSR), CD25 (7D4),T-bet (4B10) GATA-3 (TWAL), HIF-1 alpha (Mgc3), and IL-22 (IL22JOP), and human CD127 (eBioRDR5) was purchased from Invitrogen (Waltham, MA). mAbs against mouse TCR-β (H57-597), CD11b (M1/70), CD155 (TX56), KLRG1 (2F1), CD25 (PC61), NKp46 (29A1.4), Ly-6G (IA8), Gr-1 (RB6-8C5), CD80 (B7-1), CD103 (2E7), CD49b (DX5), CD45RB (C363-16A), CD90.2 (53-2.1), I-A/I-E (M5/114.15.2), CD45.2 (104), CD45 (30-F11), CD64 (X54-5/7.1), CD170/Siglec-F (S17007L), CD11c (N418), CD4 (RMA-5), CD4 (GK1.5), F4/80 (BM8), CD127(A7R34), CD11b (M1/70), Ly-6C (HK1.4), CCR6 (29-2L17), T-bet (4B10), IFN-γ (XMG1.2), GM-CSF (MP1-22E9), human CD161 (HP-3G10), CCR6 (G034E3), NKp44 (P44-8), CRTH2 (BM16), and Fluorochrome-conjugated streptavidin were purchased from Biolegend (San Diego, CA). mAb against phospho-Akt(T308) (D25E6) and phospho-S6 ribosomal protein (s235/236) (D57.2.2E) were purchased from Cell Signaling Technology (Massachusetts, USA). mAb against human CD3 was purchased from Cytec Biosciences (California, USA). mAb against human CD16 and FcεRIα were purchased from Miltenyi biotec.

    Concentration Assay:

    Article Title: Platelet extracellular vesicles drive monocyte differentiation into inflammatory foam cells in peripheral arterial disease.
    Article Snippet: .. Isolation of CD14+ CD16- monocytes used labelling of PBMCs with anti-CD16 human microbeads (Miltenyi) in MACS buffer at a concentration of 50 ml per 5 × 107 cells. .. After 20 min at 4◦C, CD16+ cells were depleted by passing them through a magnetic LD column (Miltenyi) according to manufacturer’s instructions.

    Blocking Assay:

    Article Title: SFV Replicon Vector Harbouring Porcine Epidemic Diarrhoea Virus Immunogens Delivered by Attenuated Salmonella Typhimurium Induces PEDV Neutralising Antibodies and Lactogenic Immunogenicity in BALB/c Mice
    Article Snippet: For stimulation, 2 × 10 5 viable cells/well were seeded in round-bottom 96-well plates (SPL life sciences, Republic of Korea) in complete RPMI 1640 medium and stimulated with recombinant PEDV S1-NTD (1 μg/well) and PEDV-N (1 μg/well) proteins for 72 h at 37 °C, 5% CO 2 . .. Fc Block solution was prepared using anti-CD16 (Miltenyi Biotec, Bergisch Gladbach, Germany) in FACS buffer, and cells will be incubated for 15 min at 4 °C. .. The cells were then stained with a cocktail containing anti-mouse CD3a-PE (Miltenyi Biotec, Germany), CD8-FITC (Miltenyi Biotec, Germany), and CD4-perCP-vio700 (Miltenyi Biotec, Germany) and incubated for 30 min at 4 °C in the dark.

    Article Title: SFV Replicon Vector Harbouring Porcine Epidemic Diarrhoea Virus Immunogens Delivered by Attenuated Salmonella Typhimurium Induces PEDV Neutralising Antibodies and Lactogenic Immunogenicity in BALB/c Mice.
    Article Snippet: For stimulation, 2 × 105 viable cells/well were seeded in round-bottom 96-well plates (SPL life sciences, Republic of Korea) in complete RPMI 1640 medium and stimulated with recombinant PEDV S1-NTD (1 μg/well) and PEDV-N (1 μg/well) proteins for 72 h at 37 ◦C, 5% CO2. .. Fc Block solution was prepared using anti-CD16 (Miltenyi Biotec, Bergisch Gladbach, Germany) in FACS buffer, and cells will be incubated for 15 min at 4 ◦C. .. The cells were then stained with a cocktail containing anti-mouse CD3a-PE (Miltenyi Biotec, Germany), CD8-FITC (Miltenyi Biotec, Germany), and CD4-perCP-vio700 (Miltenyi Biotec, Germany) and incubated for 30 min at 4 ◦C in the dark.

    FACS:

    Article Title: SFV Replicon Vector Harbouring Porcine Epidemic Diarrhoea Virus Immunogens Delivered by Attenuated Salmonella Typhimurium Induces PEDV Neutralising Antibodies and Lactogenic Immunogenicity in BALB/c Mice
    Article Snippet: For stimulation, 2 × 10 5 viable cells/well were seeded in round-bottom 96-well plates (SPL life sciences, Republic of Korea) in complete RPMI 1640 medium and stimulated with recombinant PEDV S1-NTD (1 μg/well) and PEDV-N (1 μg/well) proteins for 72 h at 37 °C, 5% CO 2 . .. Fc Block solution was prepared using anti-CD16 (Miltenyi Biotec, Bergisch Gladbach, Germany) in FACS buffer, and cells will be incubated for 15 min at 4 °C. .. The cells were then stained with a cocktail containing anti-mouse CD3a-PE (Miltenyi Biotec, Germany), CD8-FITC (Miltenyi Biotec, Germany), and CD4-perCP-vio700 (Miltenyi Biotec, Germany) and incubated for 30 min at 4 °C in the dark.

    Article Title: SFV Replicon Vector Harbouring Porcine Epidemic Diarrhoea Virus Immunogens Delivered by Attenuated Salmonella Typhimurium Induces PEDV Neutralising Antibodies and Lactogenic Immunogenicity in BALB/c Mice.
    Article Snippet: For stimulation, 2 × 105 viable cells/well were seeded in round-bottom 96-well plates (SPL life sciences, Republic of Korea) in complete RPMI 1640 medium and stimulated with recombinant PEDV S1-NTD (1 μg/well) and PEDV-N (1 μg/well) proteins for 72 h at 37 ◦C, 5% CO2. .. Fc Block solution was prepared using anti-CD16 (Miltenyi Biotec, Bergisch Gladbach, Germany) in FACS buffer, and cells will be incubated for 15 min at 4 ◦C. .. The cells were then stained with a cocktail containing anti-mouse CD3a-PE (Miltenyi Biotec, Germany), CD8-FITC (Miltenyi Biotec, Germany), and CD4-perCP-vio700 (Miltenyi Biotec, Germany) and incubated for 30 min at 4 ◦C in the dark.

    Staining:

    Article Title: Peripheral Blood Neutrophils and Monocytes Predict Disease Progression in Stage III Melanoma Patients Treated With Anti Programmed Cell Death Protein-1 (PD-1) Inhibitors.
    Article Snippet: PBMCs were stained (20min,+4°C) in PBS containing 1% FBS with the following antibodies: fluorescein isothiocyanate (FITC)–conjugated anti-CD14 (1:50, from Miltenyi Biotec, Bergisch Gladbach, Germany) and phycoerythrin (PE)–conjugated anti-PD-L1 (1:10, from Biolegend, CA, USA). .. Moreover, PMNs were stained (20min and +4°C) in PBS containing 1% FBS with the following antibodies: allophycocyanin (APC)–conjugated anti-CD66b (1:50, from Miltenyi Biotec, Bergisch Gladbach, Germany), peridinin chlorophyll protein (PerCP)–conjugated antiCD11b (1:50, from Miltenyi Biotec, Bergisch Gladbach, Germany), VioBlue-conjugated anti-CD193 (1:10, from Miltenyi Biotec, Bergisch Gladbach, Germany), FITC-conjugated anti-CD62L (1:50, from Miltenyi Biotec, Bergisch Gladbach, Germany), PEconjugated anti-PD-L1 (1:10, from Biolegend, CA, USA), and PEconjugated anti-CD16 (1:50, from Miltenyi Biotec, Bergisch Gladbach, Germany). .. The cells were acquired with a MACS Quant Analyzer 10 (Miltenyi Biotec, Bergisch Gladbach, Germany) and analyzedwith FlowJo v.10 software.Doublets and debris (identified according to forward and side scatter (SSC) properties), dead cells (identifiedwith the Zombie Violet Fixable Viability Kit; BioLegend, San Diego, CA, USA), and eosinophils (identified using CCR3+ exclusion gating) were excluded from the analysis.



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    During transforming growth factor β1 (TGFβ1)-induced epithelial-to-mesenchymal transition (EMT), stomatin was downregulated through <t>methylation-dependent</t> transcription mechanism. (A) Real-time polymerase chain reaction (PCR) was performed to validate the transcriptional change of stomatin identified in the microarray. **, P<0.001; (B) Two CpG islands spanning from −768 to −223 bp and from −143 to +122 bp upstream of STOM gene were identified by EMBOSS cpgplot program (upper). Schematic representation of −768/−223 and −143/+122 (marked with the grey square) CpG islands in the upstream of stomatin gene (lower). Red box marked the exon. The translation start site was position +1; (C) Measuring stomatin promoter methylation levels by Sequenom <t>MassARRAY</t> assay; (D) Sequenom MassARRAY analysis of methylation percentages of CpG sites at the stomatin promoter. The top panel showed the methylation percentages of 30 sites in human bronchial epithelial (HBE) and A549 cells. Dots with different color depths represent different methylation status. Dark dots represent high degrees of methylation. Light dots represent low degrees of methylation. The methylation percentages of 30 CpG sites were compared by two-tailed paired Student’s t test. In line graph, each dot represents an individual CpG dinucleotide. Green dots represent CpG dinucleotides in HBE cells. Red dots represent CpG sites in A549 cells. The corresponding detection sites were connected in a straight line. Real-time PCR was performed to evaluate the expression of stomatin in HBE and A549 cells (P<0.01); (E) The same sequenom MassArray and statistical analyses were also implemented to assess the methylation status of the indicated promoter region of stomatin in A549 and A549-5Aza cells (P<0.01); (F) A549 cells were exposed to TGFβ1 (5 ng/mL) for 2 d or 5 d with or without 5-Aza. Expression of DNMT3A and stomatin was analyzed by western blotting. β-actin was used as a loading control; (G) Sequenom MassArray analysis was implemented to assess the stomatin promoter methylation levels in A549, A549-TGFβ1, and A549-TGFβ1/5-Aza cells (P=0.005, P=0.004, respectively).
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    During transforming growth factor β1 (TGFβ1)-induced epithelial-to-mesenchymal transition (EMT), stomatin was downregulated through methylation-dependent transcription mechanism. (A) Real-time polymerase chain reaction (PCR) was performed to validate the transcriptional change of stomatin identified in the microarray. **, P<0.001; (B) Two CpG islands spanning from −768 to −223 bp and from −143 to +122 bp upstream of STOM gene were identified by EMBOSS cpgplot program (upper). Schematic representation of −768/−223 and −143/+122 (marked with the grey square) CpG islands in the upstream of stomatin gene (lower). Red box marked the exon. The translation start site was position +1; (C) Measuring stomatin promoter methylation levels by Sequenom MassARRAY assay; (D) Sequenom MassARRAY analysis of methylation percentages of CpG sites at the stomatin promoter. The top panel showed the methylation percentages of 30 sites in human bronchial epithelial (HBE) and A549 cells. Dots with different color depths represent different methylation status. Dark dots represent high degrees of methylation. Light dots represent low degrees of methylation. The methylation percentages of 30 CpG sites were compared by two-tailed paired Student’s t test. In line graph, each dot represents an individual CpG dinucleotide. Green dots represent CpG dinucleotides in HBE cells. Red dots represent CpG sites in A549 cells. The corresponding detection sites were connected in a straight line. Real-time PCR was performed to evaluate the expression of stomatin in HBE and A549 cells (P<0.01); (E) The same sequenom MassArray and statistical analyses were also implemented to assess the methylation status of the indicated promoter region of stomatin in A549 and A549-5Aza cells (P<0.01); (F) A549 cells were exposed to TGFβ1 (5 ng/mL) for 2 d or 5 d with or without 5-Aza. Expression of DNMT3A and stomatin was analyzed by western blotting. β-actin was used as a loading control; (G) Sequenom MassArray analysis was implemented to assess the stomatin promoter methylation levels in A549, A549-TGFβ1, and A549-TGFβ1/5-Aza cells (P=0.005, P=0.004, respectively).

    Journal: Chinese Journal of Cancer Research

    Article Title: Stomatin plays a suppressor role in non-small cell lung cancer metastasis

    doi: 10.21147/j.issn.1000-9604.2019.06.09

    Figure Lengend Snippet: During transforming growth factor β1 (TGFβ1)-induced epithelial-to-mesenchymal transition (EMT), stomatin was downregulated through methylation-dependent transcription mechanism. (A) Real-time polymerase chain reaction (PCR) was performed to validate the transcriptional change of stomatin identified in the microarray. **, P<0.001; (B) Two CpG islands spanning from −768 to −223 bp and from −143 to +122 bp upstream of STOM gene were identified by EMBOSS cpgplot program (upper). Schematic representation of −768/−223 and −143/+122 (marked with the grey square) CpG islands in the upstream of stomatin gene (lower). Red box marked the exon. The translation start site was position +1; (C) Measuring stomatin promoter methylation levels by Sequenom MassARRAY assay; (D) Sequenom MassARRAY analysis of methylation percentages of CpG sites at the stomatin promoter. The top panel showed the methylation percentages of 30 sites in human bronchial epithelial (HBE) and A549 cells. Dots with different color depths represent different methylation status. Dark dots represent high degrees of methylation. Light dots represent low degrees of methylation. The methylation percentages of 30 CpG sites were compared by two-tailed paired Student’s t test. In line graph, each dot represents an individual CpG dinucleotide. Green dots represent CpG dinucleotides in HBE cells. Red dots represent CpG sites in A549 cells. The corresponding detection sites were connected in a straight line. Real-time PCR was performed to evaluate the expression of stomatin in HBE and A549 cells (P<0.01); (E) The same sequenom MassArray and statistical analyses were also implemented to assess the methylation status of the indicated promoter region of stomatin in A549 and A549-5Aza cells (P<0.01); (F) A549 cells were exposed to TGFβ1 (5 ng/mL) for 2 d or 5 d with or without 5-Aza. Expression of DNMT3A and stomatin was analyzed by western blotting. β-actin was used as a loading control; (G) Sequenom MassArray analysis was implemented to assess the stomatin promoter methylation levels in A549, A549-TGFβ1, and A549-TGFβ1/5-Aza cells (P=0.005, P=0.004, respectively).

    Article Snippet: The methylation status of Stomatin promoter was detected using Sequenom MassARRAY quantitative methylation analysis platform (BGI tech, Shenzhen, China).

    Techniques: Methylation, Real-time Polymerase Chain Reaction, Microarray, Sequenom Massarray Assay, Two Tailed Test, Expressing, Western Blot, Control