isotype control antibody  (Millipore)


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    Name:
    Mouse IgG2a Isotype Control from murine myeloma
    Description:
    Immunoglobulin G IgG is a glycoprotein antibody that regulates immune responses such as phagocytosis and is also involved in the development of autoimmune diseases Mouse IgGs have four distinct isotypes namely IgG1 IgG2a IgG2b and IgG3 IgG1 regulates complement fixation in mice Mouse IgG2a antibodies react with anti mouse whole serum and anti mouse IgG2a The antibody product can be used as controls in immunoassays
    Catalog Number:
    m5409
    Price:
    None
    Applications:
    Mouse IgG2a Isotype Control antibodies can be used for affinity chromatography and chromatin immunoprecipitation . The product can also be used for flow cytometry.
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    Structured Review

    Millipore isotype control antibody
    Immunoglobulin G IgG is a glycoprotein antibody that regulates immune responses such as phagocytosis and is also involved in the development of autoimmune diseases Mouse IgGs have four distinct isotypes namely IgG1 IgG2a IgG2b and IgG3 IgG1 regulates complement fixation in mice Mouse IgG2a antibodies react with anti mouse whole serum and anti mouse IgG2a The antibody product can be used as controls in immunoassays
    https://www.bioz.com/result/isotype control antibody/product/Millipore
    Average 99 stars, based on 61 article reviews
    Price from $9.99 to $1999.99
    isotype control antibody - by Bioz Stars, 2021-01
    99/100 stars

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    Incubation:

    Article Title: Functional Inhibitory Siglec-6 Is Upregulated in Human Colorectal Cancer-Associated Mast Cells
    Article Snippet: .. Sections were then incubated with mouse anti-human tryptase (Abcam) or mouse IgG isotype and rabbit anti-human Siglec-6 (Sigma, HPA009084) or rabbit IgG isotype for overnight at 4°C and were subsequently detected with Alexa Fluor 488 goat anti-mouse IgG (ThermoFisher) and Alexa Fluor 594 goat anti-rabbit IgG (Invitrogen). .. Nuclei were counterstained with DAPI (Invitrogen).

    Blocking Assay:

    Article Title: Thrombospondin 1 Activates Macrophage Toll-Like 4 Receptor Pathway
    Article Snippet: .. In some experiments, prior to TSP1 treatment, a blocking peptide, a control scrambled peptide, a CD36 blocking antibody (clone FA6-152 IgG1 , from Abcam (Cambridge, MA, USA)) , or an isotype control anti-mouse IgG1 (from Sigma) was used to pre-treat the macrophages, and the above experiment was repeated. .. In addition, in some experiments, recombinant human TSP1 was incubated with polymyxin B sulfate (10 µg/ml) for 30 min and then added to macrophages.

    Labeling:

    Article Title: Phosphatase of Regenerating Liver-1 (PRL-1) Regulates Actin Dynamics During Immunological Synapse Assembly and T Cell Effector Function
    Article Snippet: .. Mouse fluorescently labeled antibodies specific for CD4 and CD45RA were obtained from Beckman Coulter (CA, USA), APC-labeled anti-CD3 from BD Pharmingen (CA, USA), rabbit anti-GFP from Life Technologies (CA, USA) and mouse anti-α tubulin and mouse IgG1 isotype control (MOPC21) from Sigma Aldrich (MO, USA). ..

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    Millipore igg1 isotype control mab
    Effects of TGFß1, with and without M7824, on NK cell phenotype and NK lytic function Panels A-C NK cells isolated from a healthy donor were incubated for 48 hours with either no treatment, TGFß1, or simultaneously treated with TGFß1 (2 ng/ml) plus isotype control <t>IgG1</t> MAb, M7824, M7824mut, or anti-PD-L1 MAb; NK cells were then phenotyped by flow cytometry. The results for NKG2D expression for three of the treatment groups are shown using NK cells from one healthy donor in dot plots. Panel A: Untreated and TGFß1-treated NK cells, Panel B: Untreated and TGFß1 plus M7824–treated NK cells, and Panel C: Untreated and TGFß1 plus anti-PD-L1 MAb–treated NK cells. Panels D-G The flow cytometry results from NK phenotyping are shown as the change after treatment, by setting the expression level of the markers at 100% for untreated NK cells. Panels H-J NK cells isolated from a healthy donor were incubated for 48 hours with no treatment, TGFß1 (2 ng/ml), TGFß1 plus control IgG, TGFß1 plus M7824, or TGFß1 plus M7824mut, and then evaluated in tumor cell lysis assays. Panel H: NK tumor cell lysis (1 μg/ml control IgG1 MAb present in assay); Panel I: ADCC of tumor cells mediated by cetuximab (1 μg/ml); Panel J: ADCC lysis of tumor cells mediated by M7824 (1 μg/ml). In studies shown in Panels H, I and J, after exposure of NK cells to TGFß1 and other treatments, cells were washed prior to use in the ADCC assays. Note different y-axes for Panel H vs. Panels I and J. T-tests were performed to compare untreated NK cells to TGFß1, and TGFß1 to all treatment groups. *** P
    Igg1 Isotype Control Mab, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/igg1 isotype control mab/product/Millipore
    Average 99 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    igg1 isotype control mab - by Bioz Stars, 2021-01
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    92
    Millipore mouse igm isotype
    Characterization of mouse <t>IgM</t> mAbs in ( A ) IFA on CHIKV-infected BHK-21 cells and ( B ) indirect virion-based ELISA. ( A ) Purified mAbs produced from immunization of CHIKV was tested at 1:100 or 1:300 dilution in PBS. Rabbit anti-CHIKV E2 polyclonal <t>IgG</t> and mouse IgM isotype antibodies serve as positive and negative control, respectively. Cell nuclei were stained with DAPI while CHIKV antigens (white arrowhead) were secondarily stained with goat anti-mouse or anti-rabbit IgG FITC. Images were captured under 10x and 100x magnification and representative images from 2 independent experiments are shown. ( B The negative control consists of wells not coated with CHIKV. Absorbance value (Abs) was measured at 450 nm. Mean abs is derived from 3 independent experiments performed in duplicates and ± SD values are shown as error bars.
    Mouse Igm Isotype, supplied by Millipore, used in various techniques. Bioz Stars score: 92/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mouse igm isotype/product/Millipore
    Average 92 stars, based on 3 article reviews
    Price from $9.99 to $1999.99
    mouse igm isotype - by Bioz Stars, 2021-01
    92/100 stars
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    99
    Millipore rabbit igg isotype control antibodies
    Post-translational histone modifications can be analyzed by FCM in zinc-fixed cells mZBF-fixed cell suspensions were stained with antibodies against NeuN, CD11b, and GFAP to identify neurons, microglia and astrocytes respectively, in the presence of antibodies against the histone mark ( A , B ) H3K27me1, ( C , D ) <t>H3K27me3,</t> or ( E , F ) pan-H3. Singlet and DAPI + gated populations were subsequently gated by cell-type, and changes in the median fluorescent intensity (MFI) of the respective histone marks were assessed. Shifts in the MFI with primary antibody relative to signal with <t>IgG</t> isotype control antibody are shown in the histograms, and averaged data ( n =4 independent samples) are shown in the bar graphs.
    Rabbit Igg Isotype Control Antibodies, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit igg isotype control antibodies/product/Millipore
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rabbit igg isotype control antibodies - by Bioz Stars, 2021-01
    99/100 stars
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    Effects of TGFß1, with and without M7824, on NK cell phenotype and NK lytic function Panels A-C NK cells isolated from a healthy donor were incubated for 48 hours with either no treatment, TGFß1, or simultaneously treated with TGFß1 (2 ng/ml) plus isotype control IgG1 MAb, M7824, M7824mut, or anti-PD-L1 MAb; NK cells were then phenotyped by flow cytometry. The results for NKG2D expression for three of the treatment groups are shown using NK cells from one healthy donor in dot plots. Panel A: Untreated and TGFß1-treated NK cells, Panel B: Untreated and TGFß1 plus M7824–treated NK cells, and Panel C: Untreated and TGFß1 plus anti-PD-L1 MAb–treated NK cells. Panels D-G The flow cytometry results from NK phenotyping are shown as the change after treatment, by setting the expression level of the markers at 100% for untreated NK cells. Panels H-J NK cells isolated from a healthy donor were incubated for 48 hours with no treatment, TGFß1 (2 ng/ml), TGFß1 plus control IgG, TGFß1 plus M7824, or TGFß1 plus M7824mut, and then evaluated in tumor cell lysis assays. Panel H: NK tumor cell lysis (1 μg/ml control IgG1 MAb present in assay); Panel I: ADCC of tumor cells mediated by cetuximab (1 μg/ml); Panel J: ADCC lysis of tumor cells mediated by M7824 (1 μg/ml). In studies shown in Panels H, I and J, after exposure of NK cells to TGFß1 and other treatments, cells were washed prior to use in the ADCC assays. Note different y-axes for Panel H vs. Panels I and J. T-tests were performed to compare untreated NK cells to TGFß1, and TGFß1 to all treatment groups. *** P

    Journal: Oncotarget

    Article Title: Analyses of functions of an anti-PD-L1/TGFβR2 bispecific fusion protein (M7824)

    doi: 10.18632/oncotarget.20680

    Figure Lengend Snippet: Effects of TGFß1, with and without M7824, on NK cell phenotype and NK lytic function Panels A-C NK cells isolated from a healthy donor were incubated for 48 hours with either no treatment, TGFß1, or simultaneously treated with TGFß1 (2 ng/ml) plus isotype control IgG1 MAb, M7824, M7824mut, or anti-PD-L1 MAb; NK cells were then phenotyped by flow cytometry. The results for NKG2D expression for three of the treatment groups are shown using NK cells from one healthy donor in dot plots. Panel A: Untreated and TGFß1-treated NK cells, Panel B: Untreated and TGFß1 plus M7824–treated NK cells, and Panel C: Untreated and TGFß1 plus anti-PD-L1 MAb–treated NK cells. Panels D-G The flow cytometry results from NK phenotyping are shown as the change after treatment, by setting the expression level of the markers at 100% for untreated NK cells. Panels H-J NK cells isolated from a healthy donor were incubated for 48 hours with no treatment, TGFß1 (2 ng/ml), TGFß1 plus control IgG, TGFß1 plus M7824, or TGFß1 plus M7824mut, and then evaluated in tumor cell lysis assays. Panel H: NK tumor cell lysis (1 μg/ml control IgG1 MAb present in assay); Panel I: ADCC of tumor cells mediated by cetuximab (1 μg/ml); Panel J: ADCC lysis of tumor cells mediated by M7824 (1 μg/ml). In studies shown in Panels H, I and J, after exposure of NK cells to TGFß1 and other treatments, cells were washed prior to use in the ADCC assays. Note different y-axes for Panel H vs. Panels I and J. T-tests were performed to compare untreated NK cells to TGFß1, and TGFß1 to all treatment groups. *** P

    Article Snippet: The M7824, M7824mut, and a matching IgG1 isotype control MAb were obtained from EMD Serono (Rockland, MA) as part of a Cooperative Research and Development Agreement (CRADA) with the National Cancer Institute, NIH.

    Techniques: Isolation, Incubation, Flow Cytometry, Cytometry, Expressing, Lysis

    Effects of M7824 on Treg suppression of CD4+ proliferation CD4 + CD25 NEG/LOW effector cells and autologous Tregs (CD4 + CD25 HIGH CD127 DIM/NEG ) were isolated from three healthy donors’ PBMCs, and used in a proliferation assay with plate-bound anti-CD3, as described in Materials and Methods. Proliferation of CD4 + effectors in the absence of Tregs was set as 100% proliferation. Panels A-C show the individual results from the three donors, and Panel D shows the average proliferation. In all panels, CD4 + effectors alone are shown as blue bars; all other bars indicate CD4 + effectors co-incubated with autologous Tregs at a 1:1 ratio with different treatments. The addition of Tregs significantly decreased the CD4 + T-cell proliferation (hatched blue bars), and this could be partially restored by treatment with M7824 (red bars) or M7824mut (hatched red bars), but not anti-PD-L1 (gray bars). A t-test was performed to compare the treatment groups to isotype IgG control for the average of all three donors. *** P

    Journal: Oncotarget

    Article Title: Analyses of functions of an anti-PD-L1/TGFβR2 bispecific fusion protein (M7824)

    doi: 10.18632/oncotarget.20680

    Figure Lengend Snippet: Effects of M7824 on Treg suppression of CD4+ proliferation CD4 + CD25 NEG/LOW effector cells and autologous Tregs (CD4 + CD25 HIGH CD127 DIM/NEG ) were isolated from three healthy donors’ PBMCs, and used in a proliferation assay with plate-bound anti-CD3, as described in Materials and Methods. Proliferation of CD4 + effectors in the absence of Tregs was set as 100% proliferation. Panels A-C show the individual results from the three donors, and Panel D shows the average proliferation. In all panels, CD4 + effectors alone are shown as blue bars; all other bars indicate CD4 + effectors co-incubated with autologous Tregs at a 1:1 ratio with different treatments. The addition of Tregs significantly decreased the CD4 + T-cell proliferation (hatched blue bars), and this could be partially restored by treatment with M7824 (red bars) or M7824mut (hatched red bars), but not anti-PD-L1 (gray bars). A t-test was performed to compare the treatment groups to isotype IgG control for the average of all three donors. *** P

    Article Snippet: The M7824, M7824mut, and a matching IgG1 isotype control MAb were obtained from EMD Serono (Rockland, MA) as part of a Cooperative Research and Development Agreement (CRADA) with the National Cancer Institute, NIH.

    Techniques: Isolation, Proliferation Assay, Incubation

    Pretreating NK cells with ALT-803 increases tumor cell lysis and ADCC mediated by M7824 NK cells were isolated from PBMCs from three cancer patients and three healthy donors; NK cells were untreated or treated (24 hours) with ALT-803 (IL-15 superagonist/IL-15RαSushi-Fc fusion complex, 25 ng/ml), and then used in 111 In-release 20-hour assays to evaluate NK tumor cell lysis (no MAb and control IgG1, 1 μg/ml), and ADCC mediated by M7824 (1 μg/ml), as described in Materials and Methods. Results from one representative cancer patient are shown using as targets four human tumor cancer cell lines: H441 (lung carcinoma, Panel A ), HCC4006 (lung carcinoma, Panel B ), MDA-MB-231 (breast carcinoma, Panel C ), and CaSki (cervical carcinoma, Panel D ). NK tumor cell lysis is shown as white squares (same results with no MAb and IgG1 control antibody, which is shown); grey diamonds denote tumor cell lysis for ALT-803–treated NK cells. ADCC mediated by NK cells plus M7824 is shown as white circles; black squares denote M7824-mediated ADCC using NK cells treated with ALT-803. Results are shown at different E:T ratios, with mean and standard deviations of triplicate wells, using NK cells from a cancer patient. NK cells from two other cancer patients and three healthy donors showed similar results. Multiple t-tests were used to compare ADCC mediated by M7824 using ALT-803–treated NK cells (black squares) vs. ADCC mediated by M7824 using untreated NK cells (white circles) at each E:T ratio, *** P

    Journal: Oncotarget

    Article Title: Analyses of functions of an anti-PD-L1/TGFβR2 bispecific fusion protein (M7824)

    doi: 10.18632/oncotarget.20680

    Figure Lengend Snippet: Pretreating NK cells with ALT-803 increases tumor cell lysis and ADCC mediated by M7824 NK cells were isolated from PBMCs from three cancer patients and three healthy donors; NK cells were untreated or treated (24 hours) with ALT-803 (IL-15 superagonist/IL-15RαSushi-Fc fusion complex, 25 ng/ml), and then used in 111 In-release 20-hour assays to evaluate NK tumor cell lysis (no MAb and control IgG1, 1 μg/ml), and ADCC mediated by M7824 (1 μg/ml), as described in Materials and Methods. Results from one representative cancer patient are shown using as targets four human tumor cancer cell lines: H441 (lung carcinoma, Panel A ), HCC4006 (lung carcinoma, Panel B ), MDA-MB-231 (breast carcinoma, Panel C ), and CaSki (cervical carcinoma, Panel D ). NK tumor cell lysis is shown as white squares (same results with no MAb and IgG1 control antibody, which is shown); grey diamonds denote tumor cell lysis for ALT-803–treated NK cells. ADCC mediated by NK cells plus M7824 is shown as white circles; black squares denote M7824-mediated ADCC using NK cells treated with ALT-803. Results are shown at different E:T ratios, with mean and standard deviations of triplicate wells, using NK cells from a cancer patient. NK cells from two other cancer patients and three healthy donors showed similar results. Multiple t-tests were used to compare ADCC mediated by M7824 using ALT-803–treated NK cells (black squares) vs. ADCC mediated by M7824 using untreated NK cells (white circles) at each E:T ratio, *** P

    Article Snippet: The M7824, M7824mut, and a matching IgG1 isotype control MAb were obtained from EMD Serono (Rockland, MA) as part of a Cooperative Research and Development Agreement (CRADA) with the National Cancer Institute, NIH.

    Techniques: Lysis, Isolation, Multiple Displacement Amplification

    ADCC of tumor cells mediated by M7824 vs. anti-PD-L1, with and without pretreatment of effector NK cells with ALT-803 NK cells were isolated from PBMCs from three healthy donors, rested untreated (Panels A, C and E ) or treated (24 hours) with ALT-803 (Panels B, D and F ), and then used in 111 In-release 20-hour assays to evaluate NK tumor cell lysis (white squares, IgG1 1 μg/ml) and ADCC mediated by M7824 and anti-PD-L1. ADCC mediated by anti-PD-L1 is shown in black circles and ADCC mediated by M7824 is shown in white circles. Only isotype control IgG1 antibody is shown, since the no MAb control overlapped. Results from one of three healthy donors are shown at different E:T ratios, with mean and standard deviations of triplicate wells using as targets H441 (lung cancer, Panels A and B), MDA-MB-231 (breast cancer, Panels C and D), and CaSki (cervical cancer, Panels E and F). NK cells from the other two donors showed similar results. Results with the use of ALT-803–treated NK cells are shown in Panels B, D, and F. Multiple t-tests were used to compare ADCC mediated by anti-PD-L1 (black circles) vs. NK lysis (white squares), and ADCC mediated by M7824 (white circles) vs. NK lysis (white squares), at each E:T ratio, *** P

    Journal: Oncotarget

    Article Title: Analyses of functions of an anti-PD-L1/TGFβR2 bispecific fusion protein (M7824)

    doi: 10.18632/oncotarget.20680

    Figure Lengend Snippet: ADCC of tumor cells mediated by M7824 vs. anti-PD-L1, with and without pretreatment of effector NK cells with ALT-803 NK cells were isolated from PBMCs from three healthy donors, rested untreated (Panels A, C and E ) or treated (24 hours) with ALT-803 (Panels B, D and F ), and then used in 111 In-release 20-hour assays to evaluate NK tumor cell lysis (white squares, IgG1 1 μg/ml) and ADCC mediated by M7824 and anti-PD-L1. ADCC mediated by anti-PD-L1 is shown in black circles and ADCC mediated by M7824 is shown in white circles. Only isotype control IgG1 antibody is shown, since the no MAb control overlapped. Results from one of three healthy donors are shown at different E:T ratios, with mean and standard deviations of triplicate wells using as targets H441 (lung cancer, Panels A and B), MDA-MB-231 (breast cancer, Panels C and D), and CaSki (cervical cancer, Panels E and F). NK cells from the other two donors showed similar results. Results with the use of ALT-803–treated NK cells are shown in Panels B, D, and F. Multiple t-tests were used to compare ADCC mediated by anti-PD-L1 (black circles) vs. NK lysis (white squares), and ADCC mediated by M7824 (white circles) vs. NK lysis (white squares), at each E:T ratio, *** P

    Article Snippet: The M7824, M7824mut, and a matching IgG1 isotype control MAb were obtained from EMD Serono (Rockland, MA) as part of a Cooperative Research and Development Agreement (CRADA) with the National Cancer Institute, NIH.

    Techniques: Isolation, Lysis, Multiple Displacement Amplification

    M7824 mediates ADCC of tumor cells employing an NK cell line (haNK) expressing the high affinity CD16 allele Tumor cell lysis mediated by haNK cells as effectors and M7824 (ADCC) were evaluated in 20-hour 111 In-release assays. haNK cells (irradiated 10 Gy 24 hours prior to the assays) were used as effector cells at different E:T ratios as indicated. Results are shown for Panel A : HCC4006 human lung carcinoma cell line, and Panel B : CaSki cervical carcinoma cell line, with IgG1 control antibody (1 μg/ml, white triangles) showing haNK lysis, and M7824 (1 μg/ml, black triangles) showing ADCC. Results shown are the averages (SD) of triplicate measurements from one of at least three comparable repeat experiments. Multiple t-tests were used to compare ADCC with haNK cell lysis at each E:T ratio. *** P

    Journal: Oncotarget

    Article Title: Analyses of functions of an anti-PD-L1/TGFβR2 bispecific fusion protein (M7824)

    doi: 10.18632/oncotarget.20680

    Figure Lengend Snippet: M7824 mediates ADCC of tumor cells employing an NK cell line (haNK) expressing the high affinity CD16 allele Tumor cell lysis mediated by haNK cells as effectors and M7824 (ADCC) were evaluated in 20-hour 111 In-release assays. haNK cells (irradiated 10 Gy 24 hours prior to the assays) were used as effector cells at different E:T ratios as indicated. Results are shown for Panel A : HCC4006 human lung carcinoma cell line, and Panel B : CaSki cervical carcinoma cell line, with IgG1 control antibody (1 μg/ml, white triangles) showing haNK lysis, and M7824 (1 μg/ml, black triangles) showing ADCC. Results shown are the averages (SD) of triplicate measurements from one of at least three comparable repeat experiments. Multiple t-tests were used to compare ADCC with haNK cell lysis at each E:T ratio. *** P

    Article Snippet: The M7824, M7824mut, and a matching IgG1 isotype control MAb were obtained from EMD Serono (Rockland, MA) as part of a Cooperative Research and Development Agreement (CRADA) with the National Cancer Institute, NIH.

    Techniques: Expressing, Lysis, Irradiation

    Effects of TGFß1 and M7824 on NK cell gene expression NK cells isolated from 2 healthy donors were incubated for 48 hours with either no treatment, or simultaneously treated with TGFß1 (2 ng/ml) plus isotype control IgG1 MAb (1 μg/ml), M7824 (1 μg/ml), M7824mut (1 μg/ml), or anti-PD-L1 MAb (0.8 μg/ml) prior to RNA extraction for NanoString analysis using the nCounter PanCancer Immune Profiling Panel of 770 immune related genes. Panel A A heatmap of the gene expression analysis is shown for the 50/770 genes (6.5%) that were up- or downregulated ≥ 3-fold in both donors after TGFß1-treatment compared to no treatment. Panel B Table showing the number of genes differentially expressed vs. untreated control.

    Journal: Oncotarget

    Article Title: Analyses of functions of an anti-PD-L1/TGFβR2 bispecific fusion protein (M7824)

    doi: 10.18632/oncotarget.20680

    Figure Lengend Snippet: Effects of TGFß1 and M7824 on NK cell gene expression NK cells isolated from 2 healthy donors were incubated for 48 hours with either no treatment, or simultaneously treated with TGFß1 (2 ng/ml) plus isotype control IgG1 MAb (1 μg/ml), M7824 (1 μg/ml), M7824mut (1 μg/ml), or anti-PD-L1 MAb (0.8 μg/ml) prior to RNA extraction for NanoString analysis using the nCounter PanCancer Immune Profiling Panel of 770 immune related genes. Panel A A heatmap of the gene expression analysis is shown for the 50/770 genes (6.5%) that were up- or downregulated ≥ 3-fold in both donors after TGFß1-treatment compared to no treatment. Panel B Table showing the number of genes differentially expressed vs. untreated control.

    Article Snippet: The M7824, M7824mut, and a matching IgG1 isotype control MAb were obtained from EMD Serono (Rockland, MA) as part of a Cooperative Research and Development Agreement (CRADA) with the National Cancer Institute, NIH.

    Techniques: Expressing, Isolation, Incubation, RNA Extraction

    M7824 can mediate ADCC NK cells were isolated from PBMCs from three cancer patients and three healthy donors, rested overnight, and then used in an 111 In-release 20-hour assay to evaluate NK lysis of tumor cells (with no MAb and with an IgG1 isotype control MAb 1 μg/ml), and ADCC mediated by M7824 (1 μg/ml), as described in Materials and Methods. Panels A and C show NK lysis (white squares) and M7824-mediated ADCC (blue circles) of the human cervical carcinoma cell line CaSki, using NK cells from one healthy donor (Panel A) and one patient with metastatic breast carcinoma (Panel C). Panel B (CaSki controls) shows that the M7824mut, a molecule encompassing a mutant anti-PD-L1 that does not bind to PD-L1 but contains the intact TGFßR2 segment, did not induce ADCC. In addition, neither M7824 nor M7824mut induced lysis of target cells in the absence of NK cells (Panel B, right side). Panels D and E show NK lysis (open squares) and M7824-mediated ADCC (blue circles) of the human lung carcinoma cell line H441 using NK cells from a healthy donor and a breast carcinoma patient, respectively. Results are shown at several E:T ratios, with mean and standard deviations of triplicate wells. All experiments have been repeated multiple times with similar results. Multiple t-tests were used to compare ADCC with NK cell lysis at each E:T ratio, *** P

    Journal: Oncotarget

    Article Title: Analyses of functions of an anti-PD-L1/TGFβR2 bispecific fusion protein (M7824)

    doi: 10.18632/oncotarget.20680

    Figure Lengend Snippet: M7824 can mediate ADCC NK cells were isolated from PBMCs from three cancer patients and three healthy donors, rested overnight, and then used in an 111 In-release 20-hour assay to evaluate NK lysis of tumor cells (with no MAb and with an IgG1 isotype control MAb 1 μg/ml), and ADCC mediated by M7824 (1 μg/ml), as described in Materials and Methods. Panels A and C show NK lysis (white squares) and M7824-mediated ADCC (blue circles) of the human cervical carcinoma cell line CaSki, using NK cells from one healthy donor (Panel A) and one patient with metastatic breast carcinoma (Panel C). Panel B (CaSki controls) shows that the M7824mut, a molecule encompassing a mutant anti-PD-L1 that does not bind to PD-L1 but contains the intact TGFßR2 segment, did not induce ADCC. In addition, neither M7824 nor M7824mut induced lysis of target cells in the absence of NK cells (Panel B, right side). Panels D and E show NK lysis (open squares) and M7824-mediated ADCC (blue circles) of the human lung carcinoma cell line H441 using NK cells from a healthy donor and a breast carcinoma patient, respectively. Results are shown at several E:T ratios, with mean and standard deviations of triplicate wells. All experiments have been repeated multiple times with similar results. Multiple t-tests were used to compare ADCC with NK cell lysis at each E:T ratio, *** P

    Article Snippet: The M7824, M7824mut, and a matching IgG1 isotype control MAb were obtained from EMD Serono (Rockland, MA) as part of a Cooperative Research and Development Agreement (CRADA) with the National Cancer Institute, NIH.

    Techniques: Isolation, Lysis, Mutagenesis

    Characterization of mouse IgM mAbs in ( A ) IFA on CHIKV-infected BHK-21 cells and ( B ) indirect virion-based ELISA. ( A ) Purified mAbs produced from immunization of CHIKV was tested at 1:100 or 1:300 dilution in PBS. Rabbit anti-CHIKV E2 polyclonal IgG and mouse IgM isotype antibodies serve as positive and negative control, respectively. Cell nuclei were stained with DAPI while CHIKV antigens (white arrowhead) were secondarily stained with goat anti-mouse or anti-rabbit IgG FITC. Images were captured under 10x and 100x magnification and representative images from 2 independent experiments are shown. ( B The negative control consists of wells not coated with CHIKV. Absorbance value (Abs) was measured at 450 nm. Mean abs is derived from 3 independent experiments performed in duplicates and ± SD values are shown as error bars.

    Journal: mAbs

    Article Title: A potent neutralizing IgM mAb targeting the N218 epitope on E2 protein protects against Chikungunya virus pathogenesis

    doi: 10.1080/19420862.2015.1083664

    Figure Lengend Snippet: Characterization of mouse IgM mAbs in ( A ) IFA on CHIKV-infected BHK-21 cells and ( B ) indirect virion-based ELISA. ( A ) Purified mAbs produced from immunization of CHIKV was tested at 1:100 or 1:300 dilution in PBS. Rabbit anti-CHIKV E2 polyclonal IgG and mouse IgM isotype antibodies serve as positive and negative control, respectively. Cell nuclei were stained with DAPI while CHIKV antigens (white arrowhead) were secondarily stained with goat anti-mouse or anti-rabbit IgG FITC. Images were captured under 10x and 100x magnification and representative images from 2 independent experiments are shown. ( B The negative control consists of wells not coated with CHIKV. Absorbance value (Abs) was measured at 450 nm. Mean abs is derived from 3 independent experiments performed in duplicates and ± SD values are shown as error bars.

    Article Snippet: After blocking at 37°C for 2h, wells were washed twice with PBST and anti-CHIKV IgM diluted to 0.1, 5, 10, 25, 50 or 100 ng, mouse mAb anti-CHIKV E2 8A4 IgG (a kind gift from Dr. Philippe Desprès, Institute Pasteur, France) or mouse IgM isotype clone GC323 (Millipore, MABC008) was added to each well.

    Techniques: Immunofluorescence, Infection, Enzyme-linked Immunosorbent Assay, Purification, Produced, Negative Control, Staining, Derivative Assay

    Evaluation of mutant CHIKV binding and neutralization by mAb 3E7b. ( A ) Confluent BHK-21 cells were infected with CHIKV-WT, CHIKV-IVT, CHIKV-E24D, CHIKV-N218D or CHIKV-D223G clones at MOI 10. At day 1 p.i., cells were fixed and co-stained with 3E7b IgM and rabbit anti-E2 polyclonal antibody as a positive control. The difference in binding was visualized with goat anti-mouse IgM FITC and anti-rabbit IgG 594 secondary antibodies. Cell nuclei were stained DAPI and images were captured under 10x and 100x magnification. White arrowhead indicates reduced 3E7b binding. Representative images from 3 independent experiments are shown. ( B ) FITC signal of 3E7b binding is expressed as a percentage over 594 signal that represents the number of infected cells, n, quantitated. Error bars represent ± SD. One-way ANOVA followed by a post-Dunnett test, with statistical significance defined as ***p

    Journal: mAbs

    Article Title: A potent neutralizing IgM mAb targeting the N218 epitope on E2 protein protects against Chikungunya virus pathogenesis

    doi: 10.1080/19420862.2015.1083664

    Figure Lengend Snippet: Evaluation of mutant CHIKV binding and neutralization by mAb 3E7b. ( A ) Confluent BHK-21 cells were infected with CHIKV-WT, CHIKV-IVT, CHIKV-E24D, CHIKV-N218D or CHIKV-D223G clones at MOI 10. At day 1 p.i., cells were fixed and co-stained with 3E7b IgM and rabbit anti-E2 polyclonal antibody as a positive control. The difference in binding was visualized with goat anti-mouse IgM FITC and anti-rabbit IgG 594 secondary antibodies. Cell nuclei were stained DAPI and images were captured under 10x and 100x magnification. White arrowhead indicates reduced 3E7b binding. Representative images from 3 independent experiments are shown. ( B ) FITC signal of 3E7b binding is expressed as a percentage over 594 signal that represents the number of infected cells, n, quantitated. Error bars represent ± SD. One-way ANOVA followed by a post-Dunnett test, with statistical significance defined as ***p

    Article Snippet: After blocking at 37°C for 2h, wells were washed twice with PBST and anti-CHIKV IgM diluted to 0.1, 5, 10, 25, 50 or 100 ng, mouse mAb anti-CHIKV E2 8A4 IgG (a kind gift from Dr. Philippe Desprès, Institute Pasteur, France) or mouse IgM isotype clone GC323 (Millipore, MABC008) was added to each well.

    Techniques: Mutagenesis, Binding Assay, Neutralization, Infection, Clone Assay, Staining, Positive Control

    Post-translational histone modifications can be analyzed by FCM in zinc-fixed cells mZBF-fixed cell suspensions were stained with antibodies against NeuN, CD11b, and GFAP to identify neurons, microglia and astrocytes respectively, in the presence of antibodies against the histone mark ( A , B ) H3K27me1, ( C , D ) H3K27me3, or ( E , F ) pan-H3. Singlet and DAPI + gated populations were subsequently gated by cell-type, and changes in the median fluorescent intensity (MFI) of the respective histone marks were assessed. Shifts in the MFI with primary antibody relative to signal with IgG isotype control antibody are shown in the histograms, and averaged data ( n =4 independent samples) are shown in the bar graphs.

    Journal: ASN NEURO

    Article Title: Cell-type-specific Jumonji histone demethylase gene expression in the healthy rat CNS: detection by a novel flow cytometry method

    doi: 10.1042/AN20130050

    Figure Lengend Snippet: Post-translational histone modifications can be analyzed by FCM in zinc-fixed cells mZBF-fixed cell suspensions were stained with antibodies against NeuN, CD11b, and GFAP to identify neurons, microglia and astrocytes respectively, in the presence of antibodies against the histone mark ( A , B ) H3K27me1, ( C , D ) H3K27me3, or ( E , F ) pan-H3. Singlet and DAPI + gated populations were subsequently gated by cell-type, and changes in the median fluorescent intensity (MFI) of the respective histone marks were assessed. Shifts in the MFI with primary antibody relative to signal with IgG isotype control antibody are shown in the histograms, and averaged data ( n =4 independent samples) are shown in the bar graphs.

    Article Snippet: NeuN, EAAT2 (excitatory amino-acid transporter 2) [GLT-1 (glutamate transporter 1)], H3K27me3 (trimethylated H3K27), H3 and the rabbit IgG isotype control antibodies were purchased from Millipore.

    Techniques: Staining