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rat anti mouse p2x7 monoclonal antibody mab  (Enzo Biochem)

 
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    Enzo Biochem rat anti mouse p2x7 monoclonal antibody mab
    <t>P2X7</t> antagonists inhibit ATP-induced ethidium + uptake into J774 macrophage cells in a concentration-dependent manner. (a and b) J774 cells in NaCl medium were incubated with (a and b) 25 μ M ethidium + or (b) 1 μ M YO-PRO-1 2+ in the absence (basal) or presence of (a and b) 1 mM ATP or (a) 0.1 mM BzATP at 37°C for 5 min. (c) Cells in NaCl medium were preincubated with Brilliant Blue G (BBG), A438079, AZ10606120, and AZ11645373 (as indicated) at 37°C for 15 min. Ethidium + (25 μ M) was then added, and cells were incubated in the absence or presence of 1 mM ATP at 37°C for 5 min. (a–c) Incubations were stopped by the addition of MgCl 2 medium and centrifugation. Mean fluorescence intensity (MFI) of fluorescent cation uptake (pore formation) was determined by flow cytometry. (a and b) Results shown as means ± SD, n = 3; *** P < 0.001 compared to corresponding basal; ††† P < 0.001 compared to corresponding ATP. (c) Curves presented as a percentage of the maximal ATP-induced ethidium + uptake and expressed as the mean ± SD, n = 3-4.
    Rat Anti Mouse P2x7 Monoclonal Antibody Mab, supplied by Enzo Biochem, 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/rat anti mouse p2x7 monoclonal antibody mab/product/Enzo Biochem
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rat anti mouse p2x7 monoclonal antibody mab - by Bioz Stars, 2024-10
    86/100 stars

    Images

    1) Product Images from "P2X7 Receptor Activation Induces Reactive Oxygen Species Formation and Cell Death in Murine EOC13 Microglia"

    Article Title: P2X7 Receptor Activation Induces Reactive Oxygen Species Formation and Cell Death in Murine EOC13 Microglia

    Journal: Mediators of Inflammation

    doi: 10.1155/2013/271813

    P2X7 antagonists inhibit ATP-induced ethidium + uptake into J774 macrophage cells in a concentration-dependent manner. (a and b) J774 cells in NaCl medium were incubated with (a and b) 25 μ M ethidium + or (b) 1 μ M YO-PRO-1 2+ in the absence (basal) or presence of (a and b) 1 mM ATP or (a) 0.1 mM BzATP at 37°C for 5 min. (c) Cells in NaCl medium were preincubated with Brilliant Blue G (BBG), A438079, AZ10606120, and AZ11645373 (as indicated) at 37°C for 15 min. Ethidium + (25 μ M) was then added, and cells were incubated in the absence or presence of 1 mM ATP at 37°C for 5 min. (a–c) Incubations were stopped by the addition of MgCl 2 medium and centrifugation. Mean fluorescence intensity (MFI) of fluorescent cation uptake (pore formation) was determined by flow cytometry. (a and b) Results shown as means ± SD, n = 3; *** P < 0.001 compared to corresponding basal; ††† P < 0.001 compared to corresponding ATP. (c) Curves presented as a percentage of the maximal ATP-induced ethidium + uptake and expressed as the mean ± SD, n = 3-4.
    Figure Legend Snippet: P2X7 antagonists inhibit ATP-induced ethidium + uptake into J774 macrophage cells in a concentration-dependent manner. (a and b) J774 cells in NaCl medium were incubated with (a and b) 25 μ M ethidium + or (b) 1 μ M YO-PRO-1 2+ in the absence (basal) or presence of (a and b) 1 mM ATP or (a) 0.1 mM BzATP at 37°C for 5 min. (c) Cells in NaCl medium were preincubated with Brilliant Blue G (BBG), A438079, AZ10606120, and AZ11645373 (as indicated) at 37°C for 15 min. Ethidium + (25 μ M) was then added, and cells were incubated in the absence or presence of 1 mM ATP at 37°C for 5 min. (a–c) Incubations were stopped by the addition of MgCl 2 medium and centrifugation. Mean fluorescence intensity (MFI) of fluorescent cation uptake (pore formation) was determined by flow cytometry. (a and b) Results shown as means ± SD, n = 3; *** P < 0.001 compared to corresponding basal; ††† P < 0.001 compared to corresponding ATP. (c) Curves presented as a percentage of the maximal ATP-induced ethidium + uptake and expressed as the mean ± SD, n = 3-4.

    Techniques Used: Concentration Assay, Incubation, Centrifugation, Fluorescence, Flow Cytometry

    EOC13 microglial cells express P2X7. (a) RNA from EOC13 and J774 cells was amplified by RT-PCR using primers for P2X7. Water in place of RNA was included as a negative control in the PCR reaction. PCR products were separated and visualised with ethidium bromide staining. (b) EOC13 and J774 cell lysates were separated by SDS-PAGE, transferred to nitrocellulose, and probed with an anti-P2X7 Ab. (c) EOC13 and J774 cells were labelled with an anti-P2X7 (solid line) or isotype control (shaded) mAb and then with APC-conjugated anti-IgG Ab and 7AAD (to exclude dead cells). Relative P2X7 expression (mean fluorescence intensity) was determined by flow cytometry. (d) Fixed and permeabilised EOC13 and J774 cells were labelled with an anti-P2X7 Ab and then with Cy3-conjugated anti-IgG Ab. P2X7 (top panels) and phase contrast (bottom panels) images were assessed by confocal microscopy. Bars represent 10 μ m. (a–d) Results are representative of 2-3 experiments.
    Figure Legend Snippet: EOC13 microglial cells express P2X7. (a) RNA from EOC13 and J774 cells was amplified by RT-PCR using primers for P2X7. Water in place of RNA was included as a negative control in the PCR reaction. PCR products were separated and visualised with ethidium bromide staining. (b) EOC13 and J774 cell lysates were separated by SDS-PAGE, transferred to nitrocellulose, and probed with an anti-P2X7 Ab. (c) EOC13 and J774 cells were labelled with an anti-P2X7 (solid line) or isotype control (shaded) mAb and then with APC-conjugated anti-IgG Ab and 7AAD (to exclude dead cells). Relative P2X7 expression (mean fluorescence intensity) was determined by flow cytometry. (d) Fixed and permeabilised EOC13 and J774 cells were labelled with an anti-P2X7 Ab and then with Cy3-conjugated anti-IgG Ab. P2X7 (top panels) and phase contrast (bottom panels) images were assessed by confocal microscopy. Bars represent 10 μ m. (a–d) Results are representative of 2-3 experiments.

    Techniques Used: Amplification, Reverse Transcription Polymerase Chain Reaction, Negative Control, Staining, SDS Page, Expressing, Fluorescence, Flow Cytometry, Confocal Microscopy

    EOC13 microglial cells express functional P2X7. (a and b) EOC13 cells in NaCl medium were incubated with 25 μ M ethidium + in the absence (basal) or presence of (a) 1 mM ATP, 0.1 mM BzATP, or (b) varying concentrations of ATP (as indicated) at 37°C for 5 min. (c and d) Cells in NaCl medium were preincubated in the absence (control) or presence of (c) 30 μ M Brilliant Blue G (BBG), 100 μ M A438079, 30 μ M AZ11645373, or (c and d) 10 μ M AZ10606120 at 37°C for 15 min. (c) Ethidium + (25 μ M) or (d) YO-PRO-1 2+ (1 μ M) was then added, and (c and d) cells were incubated in the absence (basal) or presence of 1 mM ATP at 37°C for 5 min. (a–d) Incubations were stopped by the addition of MgCl 2 medium and centrifugation. Mean fluorescence intensity (MFI) of fluorescent cation uptake (pore formation) was determined by flow cytometry. (a, c, and d) Results shown as means ± SD, n = 3; *** P < 0.001 compared to corresponding basal; ††† P < 0.001 compared to corresponding ATP in the absence of antagonist. (b) Curve presented as a percentage of the maximal ATP-induced ethidium + uptake and expressed as the mean ± SD, n = 3.
    Figure Legend Snippet: EOC13 microglial cells express functional P2X7. (a and b) EOC13 cells in NaCl medium were incubated with 25 μ M ethidium + in the absence (basal) or presence of (a) 1 mM ATP, 0.1 mM BzATP, or (b) varying concentrations of ATP (as indicated) at 37°C for 5 min. (c and d) Cells in NaCl medium were preincubated in the absence (control) or presence of (c) 30 μ M Brilliant Blue G (BBG), 100 μ M A438079, 30 μ M AZ11645373, or (c and d) 10 μ M AZ10606120 at 37°C for 15 min. (c) Ethidium + (25 μ M) or (d) YO-PRO-1 2+ (1 μ M) was then added, and (c and d) cells were incubated in the absence (basal) or presence of 1 mM ATP at 37°C for 5 min. (a–d) Incubations were stopped by the addition of MgCl 2 medium and centrifugation. Mean fluorescence intensity (MFI) of fluorescent cation uptake (pore formation) was determined by flow cytometry. (a, c, and d) Results shown as means ± SD, n = 3; *** P < 0.001 compared to corresponding basal; ††† P < 0.001 compared to corresponding ATP in the absence of antagonist. (b) Curve presented as a percentage of the maximal ATP-induced ethidium + uptake and expressed as the mean ± SD, n = 3.

    Techniques Used: Functional Assay, Incubation, Centrifugation, Fluorescence, Flow Cytometry

    P2X7 activation induces ROS formation in EOC13 microglial cells. (Left panels) Adherent DCF-loaded EOC13 cells or (right panels) suspended EOC13 cells in (a) NaCl medium containing 1 mM Ca 2+ (preincubated in the absence (control) or presence of 10 μ M AZ10606120 at 37°C for 15 min), (b) NaCl medium in the absence (control) or presence of 1 mM Ca 2+ , (c) NaCl medium in the absence (control) or presence of 100 μ M EGTA, or (d) NaCl or KCl medium were (a–d) incubated in the absence (basal) or presence of 575 μ M ATP 4− (2 mM or 1.4 mM ATP as explained in Section 2.8) at 37°C for (left panels) 15 min or (right panels) 5 min in the presence of 25 μ M ethidium + . (a–d) Incubations were stopped by the addition of MgCl 2 medium and centrifugation. Mean fluorescence intensities (MFI) of (left panels) DCF (ROS formation) or (right panels) ethidium + uptake (pore formation) were determined by flow cytometry and results shown as means ± SD, n = 3; *** P < 0.001 or ** P < 0.01 compared to corresponding basal; ††† P < 0.001 compared to corresponding ATP.
    Figure Legend Snippet: P2X7 activation induces ROS formation in EOC13 microglial cells. (Left panels) Adherent DCF-loaded EOC13 cells or (right panels) suspended EOC13 cells in (a) NaCl medium containing 1 mM Ca 2+ (preincubated in the absence (control) or presence of 10 μ M AZ10606120 at 37°C for 15 min), (b) NaCl medium in the absence (control) or presence of 1 mM Ca 2+ , (c) NaCl medium in the absence (control) or presence of 100 μ M EGTA, or (d) NaCl or KCl medium were (a–d) incubated in the absence (basal) or presence of 575 μ M ATP 4− (2 mM or 1.4 mM ATP as explained in Section 2.8) at 37°C for (left panels) 15 min or (right panels) 5 min in the presence of 25 μ M ethidium + . (a–d) Incubations were stopped by the addition of MgCl 2 medium and centrifugation. Mean fluorescence intensities (MFI) of (left panels) DCF (ROS formation) or (right panels) ethidium + uptake (pore formation) were determined by flow cytometry and results shown as means ± SD, n = 3; *** P < 0.001 or ** P < 0.01 compared to corresponding basal; ††† P < 0.001 compared to corresponding ATP.

    Techniques Used: Activation Assay, Incubation, Centrifugation, Fluorescence, Flow Cytometry

    The ROS scavenger NAC inhibits P2X7-induced ROS and pore formation in EOC13 microglial cells. (a and c) Adherent DCF-loaded EOC13 cells or (b) suspended EOC13 cells in NaCl medium were preincubated in the absence (control) or presence of 40 mM NAC at 37°C for 30 min and then in the absence (basal) or presence of 1.4 mM ATP for (a and c) 15 min or (b) 5 min in the presence of 25 μ M ethidium + . (a–c) Incubations were stopped by the addition of MgCl 2 medium and (a and b) centrifugation. (a and b) Mean fluorescence intensities (MFI) of (a) DCF (ROS formation) or (b) ethidium + uptake (pore formation) were determined by flow cytometry and results shown as means ± SD, n = 3; *** P < 0.001 compared to corresponding basal; ††† P < 0.001 compared to corresponding ATP in the absence of NAC. (c) DIC images of cell morphology were acquired by microscopy. Bars represent 20 μ m. Results are representative of 2 experiments.
    Figure Legend Snippet: The ROS scavenger NAC inhibits P2X7-induced ROS and pore formation in EOC13 microglial cells. (a and c) Adherent DCF-loaded EOC13 cells or (b) suspended EOC13 cells in NaCl medium were preincubated in the absence (control) or presence of 40 mM NAC at 37°C for 30 min and then in the absence (basal) or presence of 1.4 mM ATP for (a and c) 15 min or (b) 5 min in the presence of 25 μ M ethidium + . (a–c) Incubations were stopped by the addition of MgCl 2 medium and (a and b) centrifugation. (a and b) Mean fluorescence intensities (MFI) of (a) DCF (ROS formation) or (b) ethidium + uptake (pore formation) were determined by flow cytometry and results shown as means ± SD, n = 3; *** P < 0.001 compared to corresponding basal; ††† P < 0.001 compared to corresponding ATP in the absence of NAC. (c) DIC images of cell morphology were acquired by microscopy. Bars represent 20 μ m. Results are representative of 2 experiments.

    Techniques Used: Centrifugation, Fluorescence, Flow Cytometry, Microscopy

    P2X7 activation induces NO formation in EOC13 microglial cells. Adherent DAF-FM DA-loaded EOC13 cells in NaCl medium were preincubated in the absence (control) or presence of 10 μ M AZ10606120 at 37°C for 15 min and then in the absence (basal) or presence of 1.4 mM ATP for 15 min. Incubations were stopped by the addition of MgCl 2 medium and centrifugation. Mean fluorescence intensities (MFI) of benzotriazole (NO formation) were determined by flow cytometry and results shown as means ± SD, n = 3; *** P < 0.001 compared to corresponding basal; ††† P < 0.001 compared to corresponding ATP.
    Figure Legend Snippet: P2X7 activation induces NO formation in EOC13 microglial cells. Adherent DAF-FM DA-loaded EOC13 cells in NaCl medium were preincubated in the absence (control) or presence of 10 μ M AZ10606120 at 37°C for 15 min and then in the absence (basal) or presence of 1.4 mM ATP for 15 min. Incubations were stopped by the addition of MgCl 2 medium and centrifugation. Mean fluorescence intensities (MFI) of benzotriazole (NO formation) were determined by flow cytometry and results shown as means ± SD, n = 3; *** P < 0.001 compared to corresponding basal; ††† P < 0.001 compared to corresponding ATP.

    Techniques Used: Activation Assay, Centrifugation, Fluorescence, Flow Cytometry

    P2X7 activation induces cell death in EOC13 microglial cells. (a) Adherent EOC13 cells in complete DMEM medium were incubated in the absence or presence of varying concentrations of ATP (as indicated) at 37°C for 24 h. (b) Adherent cells in complete DMEM medium were preincubated in the absence or presence of 10 μ M AZ10606120 at 37°C for 15 min and then in the absence or presence of 2 mM ATP for 24 h. (e and f) Adherent cells in complete DMEM medium were incubated in the absence or presence of 40 mM NAC at 37°C for 90 min and incubated in the absence or presence of 2 mM ATP for the final (e) 15–60 min or (f) 45 min (of the 90 min incubation), and then the medium replaced with fresh complete DMEM medium for 24 h. (a, b, and e) Cells were harvested, labelled with Annexin-V-Fluorescein and 7AAD, and the percentage of Annexin-V − /7AAD + , Annexin-V + /7AAD − , and Annexin-V + /7AAD + cells (together representing total cell death) determined by flow cytometry. (f) DIC images of cell morphology were acquired by microscopy. Bars represent 20 μ m. (c) Adherent DCF-loaded cells in complete DMEM medium were incubated in the absence (basal) or presence of varying concentrations of ATP (as indicated) at 37°C for 15 min. (d) Adherent DCF-loaded cells in complete DMEM medium were preincubated in the absence (control) or presence of 10 μ M AZ10606120 at 37°C for 15 min and then in the absence (basal) or presence of 2 mM ATP for 15 min. (c and d) Incubations were stopped by the addition of MgCl 2 medium and centrifugation. Mean fluorescence intensity (MFI) of DCF (ROS formation) was determined by flow cytometry. Results shown as (a) dot plots of one representative set of data demonstrating the quadrant markers and (a–e) means ± SD, n = 3; *** P < 0.001 or * P < 0.05 compared to (a and c) 0 mM ATP, (b and d) corresponding basal, or (e) corresponding 0 min ATP; ††† P < 0.001 compared to corresponding ATP in the absence of (b and d) AZ10606120 or (e) NAC.
    Figure Legend Snippet: P2X7 activation induces cell death in EOC13 microglial cells. (a) Adherent EOC13 cells in complete DMEM medium were incubated in the absence or presence of varying concentrations of ATP (as indicated) at 37°C for 24 h. (b) Adherent cells in complete DMEM medium were preincubated in the absence or presence of 10 μ M AZ10606120 at 37°C for 15 min and then in the absence or presence of 2 mM ATP for 24 h. (e and f) Adherent cells in complete DMEM medium were incubated in the absence or presence of 40 mM NAC at 37°C for 90 min and incubated in the absence or presence of 2 mM ATP for the final (e) 15–60 min or (f) 45 min (of the 90 min incubation), and then the medium replaced with fresh complete DMEM medium for 24 h. (a, b, and e) Cells were harvested, labelled with Annexin-V-Fluorescein and 7AAD, and the percentage of Annexin-V − /7AAD + , Annexin-V + /7AAD − , and Annexin-V + /7AAD + cells (together representing total cell death) determined by flow cytometry. (f) DIC images of cell morphology were acquired by microscopy. Bars represent 20 μ m. (c) Adherent DCF-loaded cells in complete DMEM medium were incubated in the absence (basal) or presence of varying concentrations of ATP (as indicated) at 37°C for 15 min. (d) Adherent DCF-loaded cells in complete DMEM medium were preincubated in the absence (control) or presence of 10 μ M AZ10606120 at 37°C for 15 min and then in the absence (basal) or presence of 2 mM ATP for 15 min. (c and d) Incubations were stopped by the addition of MgCl 2 medium and centrifugation. Mean fluorescence intensity (MFI) of DCF (ROS formation) was determined by flow cytometry. Results shown as (a) dot plots of one representative set of data demonstrating the quadrant markers and (a–e) means ± SD, n = 3; *** P < 0.001 or * P < 0.05 compared to (a and c) 0 mM ATP, (b and d) corresponding basal, or (e) corresponding 0 min ATP; ††† P < 0.001 compared to corresponding ATP in the absence of (b and d) AZ10606120 or (e) NAC.

    Techniques Used: Activation Assay, Incubation, Flow Cytometry, Microscopy, Centrifugation, Fluorescence



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    P2X7 antagonists inhibit ATP-induced ethidium + uptake into J774 macrophage cells in a concentration-dependent manner. (a and b) J774 cells in NaCl medium were incubated with (a and b) 25 μ M ethidium + or (b) 1 μ M YO-PRO-1 2+ in the absence (basal) or presence of (a and b) 1 mM ATP or (a) 0.1 mM BzATP at 37°C for 5 min. (c) Cells in NaCl medium were preincubated with Brilliant Blue G (BBG), A438079, AZ10606120, and AZ11645373 (as indicated) at 37°C for 15 min. Ethidium + (25 μ M) was then added, and cells were incubated in the absence or presence of 1 mM ATP at 37°C for 5 min. (a–c) Incubations were stopped by the addition of MgCl 2 medium and centrifugation. Mean fluorescence intensity (MFI) of fluorescent cation uptake (pore formation) was determined by flow cytometry. (a and b) Results shown as means ± SD, n = 3; *** P < 0.001 compared to corresponding basal; ††† P < 0.001 compared to corresponding ATP. (c) Curves presented as a percentage of the maximal ATP-induced ethidium + uptake and expressed as the mean ± SD, n = 3-4.

    Journal: Mediators of Inflammation

    Article Title: P2X7 Receptor Activation Induces Reactive Oxygen Species Formation and Cell Death in Murine EOC13 Microglia

    doi: 10.1155/2013/271813

    Figure Lengend Snippet: P2X7 antagonists inhibit ATP-induced ethidium + uptake into J774 macrophage cells in a concentration-dependent manner. (a and b) J774 cells in NaCl medium were incubated with (a and b) 25 μ M ethidium + or (b) 1 μ M YO-PRO-1 2+ in the absence (basal) or presence of (a and b) 1 mM ATP or (a) 0.1 mM BzATP at 37°C for 5 min. (c) Cells in NaCl medium were preincubated with Brilliant Blue G (BBG), A438079, AZ10606120, and AZ11645373 (as indicated) at 37°C for 15 min. Ethidium + (25 μ M) was then added, and cells were incubated in the absence or presence of 1 mM ATP at 37°C for 5 min. (a–c) Incubations were stopped by the addition of MgCl 2 medium and centrifugation. Mean fluorescence intensity (MFI) of fluorescent cation uptake (pore formation) was determined by flow cytometry. (a and b) Results shown as means ± SD, n = 3; *** P < 0.001 compared to corresponding basal; ††† P < 0.001 compared to corresponding ATP. (c) Curves presented as a percentage of the maximal ATP-induced ethidium + uptake and expressed as the mean ± SD, n = 3-4.

    Article Snippet: Rat anti-mouse P2X7 monoclonal antibody (mAb) (clone HANO43) was from Enzo Life Sciences.

    Techniques: Concentration Assay, Incubation, Centrifugation, Fluorescence, Flow Cytometry

    EOC13 microglial cells express P2X7. (a) RNA from EOC13 and J774 cells was amplified by RT-PCR using primers for P2X7. Water in place of RNA was included as a negative control in the PCR reaction. PCR products were separated and visualised with ethidium bromide staining. (b) EOC13 and J774 cell lysates were separated by SDS-PAGE, transferred to nitrocellulose, and probed with an anti-P2X7 Ab. (c) EOC13 and J774 cells were labelled with an anti-P2X7 (solid line) or isotype control (shaded) mAb and then with APC-conjugated anti-IgG Ab and 7AAD (to exclude dead cells). Relative P2X7 expression (mean fluorescence intensity) was determined by flow cytometry. (d) Fixed and permeabilised EOC13 and J774 cells were labelled with an anti-P2X7 Ab and then with Cy3-conjugated anti-IgG Ab. P2X7 (top panels) and phase contrast (bottom panels) images were assessed by confocal microscopy. Bars represent 10 μ m. (a–d) Results are representative of 2-3 experiments.

    Journal: Mediators of Inflammation

    Article Title: P2X7 Receptor Activation Induces Reactive Oxygen Species Formation and Cell Death in Murine EOC13 Microglia

    doi: 10.1155/2013/271813

    Figure Lengend Snippet: EOC13 microglial cells express P2X7. (a) RNA from EOC13 and J774 cells was amplified by RT-PCR using primers for P2X7. Water in place of RNA was included as a negative control in the PCR reaction. PCR products were separated and visualised with ethidium bromide staining. (b) EOC13 and J774 cell lysates were separated by SDS-PAGE, transferred to nitrocellulose, and probed with an anti-P2X7 Ab. (c) EOC13 and J774 cells were labelled with an anti-P2X7 (solid line) or isotype control (shaded) mAb and then with APC-conjugated anti-IgG Ab and 7AAD (to exclude dead cells). Relative P2X7 expression (mean fluorescence intensity) was determined by flow cytometry. (d) Fixed and permeabilised EOC13 and J774 cells were labelled with an anti-P2X7 Ab and then with Cy3-conjugated anti-IgG Ab. P2X7 (top panels) and phase contrast (bottom panels) images were assessed by confocal microscopy. Bars represent 10 μ m. (a–d) Results are representative of 2-3 experiments.

    Article Snippet: Rat anti-mouse P2X7 monoclonal antibody (mAb) (clone HANO43) was from Enzo Life Sciences.

    Techniques: Amplification, Reverse Transcription Polymerase Chain Reaction, Negative Control, Staining, SDS Page, Expressing, Fluorescence, Flow Cytometry, Confocal Microscopy

    EOC13 microglial cells express functional P2X7. (a and b) EOC13 cells in NaCl medium were incubated with 25 μ M ethidium + in the absence (basal) or presence of (a) 1 mM ATP, 0.1 mM BzATP, or (b) varying concentrations of ATP (as indicated) at 37°C for 5 min. (c and d) Cells in NaCl medium were preincubated in the absence (control) or presence of (c) 30 μ M Brilliant Blue G (BBG), 100 μ M A438079, 30 μ M AZ11645373, or (c and d) 10 μ M AZ10606120 at 37°C for 15 min. (c) Ethidium + (25 μ M) or (d) YO-PRO-1 2+ (1 μ M) was then added, and (c and d) cells were incubated in the absence (basal) or presence of 1 mM ATP at 37°C for 5 min. (a–d) Incubations were stopped by the addition of MgCl 2 medium and centrifugation. Mean fluorescence intensity (MFI) of fluorescent cation uptake (pore formation) was determined by flow cytometry. (a, c, and d) Results shown as means ± SD, n = 3; *** P < 0.001 compared to corresponding basal; ††† P < 0.001 compared to corresponding ATP in the absence of antagonist. (b) Curve presented as a percentage of the maximal ATP-induced ethidium + uptake and expressed as the mean ± SD, n = 3.

    Journal: Mediators of Inflammation

    Article Title: P2X7 Receptor Activation Induces Reactive Oxygen Species Formation and Cell Death in Murine EOC13 Microglia

    doi: 10.1155/2013/271813

    Figure Lengend Snippet: EOC13 microglial cells express functional P2X7. (a and b) EOC13 cells in NaCl medium were incubated with 25 μ M ethidium + in the absence (basal) or presence of (a) 1 mM ATP, 0.1 mM BzATP, or (b) varying concentrations of ATP (as indicated) at 37°C for 5 min. (c and d) Cells in NaCl medium were preincubated in the absence (control) or presence of (c) 30 μ M Brilliant Blue G (BBG), 100 μ M A438079, 30 μ M AZ11645373, or (c and d) 10 μ M AZ10606120 at 37°C for 15 min. (c) Ethidium + (25 μ M) or (d) YO-PRO-1 2+ (1 μ M) was then added, and (c and d) cells were incubated in the absence (basal) or presence of 1 mM ATP at 37°C for 5 min. (a–d) Incubations were stopped by the addition of MgCl 2 medium and centrifugation. Mean fluorescence intensity (MFI) of fluorescent cation uptake (pore formation) was determined by flow cytometry. (a, c, and d) Results shown as means ± SD, n = 3; *** P < 0.001 compared to corresponding basal; ††† P < 0.001 compared to corresponding ATP in the absence of antagonist. (b) Curve presented as a percentage of the maximal ATP-induced ethidium + uptake and expressed as the mean ± SD, n = 3.

    Article Snippet: Rat anti-mouse P2X7 monoclonal antibody (mAb) (clone HANO43) was from Enzo Life Sciences.

    Techniques: Functional Assay, Incubation, Centrifugation, Fluorescence, Flow Cytometry

    P2X7 activation induces ROS formation in EOC13 microglial cells. (Left panels) Adherent DCF-loaded EOC13 cells or (right panels) suspended EOC13 cells in (a) NaCl medium containing 1 mM Ca 2+ (preincubated in the absence (control) or presence of 10 μ M AZ10606120 at 37°C for 15 min), (b) NaCl medium in the absence (control) or presence of 1 mM Ca 2+ , (c) NaCl medium in the absence (control) or presence of 100 μ M EGTA, or (d) NaCl or KCl medium were (a–d) incubated in the absence (basal) or presence of 575 μ M ATP 4− (2 mM or 1.4 mM ATP as explained in Section 2.8) at 37°C for (left panels) 15 min or (right panels) 5 min in the presence of 25 μ M ethidium + . (a–d) Incubations were stopped by the addition of MgCl 2 medium and centrifugation. Mean fluorescence intensities (MFI) of (left panels) DCF (ROS formation) or (right panels) ethidium + uptake (pore formation) were determined by flow cytometry and results shown as means ± SD, n = 3; *** P < 0.001 or ** P < 0.01 compared to corresponding basal; ††† P < 0.001 compared to corresponding ATP.

    Journal: Mediators of Inflammation

    Article Title: P2X7 Receptor Activation Induces Reactive Oxygen Species Formation and Cell Death in Murine EOC13 Microglia

    doi: 10.1155/2013/271813

    Figure Lengend Snippet: P2X7 activation induces ROS formation in EOC13 microglial cells. (Left panels) Adherent DCF-loaded EOC13 cells or (right panels) suspended EOC13 cells in (a) NaCl medium containing 1 mM Ca 2+ (preincubated in the absence (control) or presence of 10 μ M AZ10606120 at 37°C for 15 min), (b) NaCl medium in the absence (control) or presence of 1 mM Ca 2+ , (c) NaCl medium in the absence (control) or presence of 100 μ M EGTA, or (d) NaCl or KCl medium were (a–d) incubated in the absence (basal) or presence of 575 μ M ATP 4− (2 mM or 1.4 mM ATP as explained in Section 2.8) at 37°C for (left panels) 15 min or (right panels) 5 min in the presence of 25 μ M ethidium + . (a–d) Incubations were stopped by the addition of MgCl 2 medium and centrifugation. Mean fluorescence intensities (MFI) of (left panels) DCF (ROS formation) or (right panels) ethidium + uptake (pore formation) were determined by flow cytometry and results shown as means ± SD, n = 3; *** P < 0.001 or ** P < 0.01 compared to corresponding basal; ††† P < 0.001 compared to corresponding ATP.

    Article Snippet: Rat anti-mouse P2X7 monoclonal antibody (mAb) (clone HANO43) was from Enzo Life Sciences.

    Techniques: Activation Assay, Incubation, Centrifugation, Fluorescence, Flow Cytometry

    The ROS scavenger NAC inhibits P2X7-induced ROS and pore formation in EOC13 microglial cells. (a and c) Adherent DCF-loaded EOC13 cells or (b) suspended EOC13 cells in NaCl medium were preincubated in the absence (control) or presence of 40 mM NAC at 37°C for 30 min and then in the absence (basal) or presence of 1.4 mM ATP for (a and c) 15 min or (b) 5 min in the presence of 25 μ M ethidium + . (a–c) Incubations were stopped by the addition of MgCl 2 medium and (a and b) centrifugation. (a and b) Mean fluorescence intensities (MFI) of (a) DCF (ROS formation) or (b) ethidium + uptake (pore formation) were determined by flow cytometry and results shown as means ± SD, n = 3; *** P < 0.001 compared to corresponding basal; ††† P < 0.001 compared to corresponding ATP in the absence of NAC. (c) DIC images of cell morphology were acquired by microscopy. Bars represent 20 μ m. Results are representative of 2 experiments.

    Journal: Mediators of Inflammation

    Article Title: P2X7 Receptor Activation Induces Reactive Oxygen Species Formation and Cell Death in Murine EOC13 Microglia

    doi: 10.1155/2013/271813

    Figure Lengend Snippet: The ROS scavenger NAC inhibits P2X7-induced ROS and pore formation in EOC13 microglial cells. (a and c) Adherent DCF-loaded EOC13 cells or (b) suspended EOC13 cells in NaCl medium were preincubated in the absence (control) or presence of 40 mM NAC at 37°C for 30 min and then in the absence (basal) or presence of 1.4 mM ATP for (a and c) 15 min or (b) 5 min in the presence of 25 μ M ethidium + . (a–c) Incubations were stopped by the addition of MgCl 2 medium and (a and b) centrifugation. (a and b) Mean fluorescence intensities (MFI) of (a) DCF (ROS formation) or (b) ethidium + uptake (pore formation) were determined by flow cytometry and results shown as means ± SD, n = 3; *** P < 0.001 compared to corresponding basal; ††† P < 0.001 compared to corresponding ATP in the absence of NAC. (c) DIC images of cell morphology were acquired by microscopy. Bars represent 20 μ m. Results are representative of 2 experiments.

    Article Snippet: Rat anti-mouse P2X7 monoclonal antibody (mAb) (clone HANO43) was from Enzo Life Sciences.

    Techniques: Centrifugation, Fluorescence, Flow Cytometry, Microscopy

    P2X7 activation induces NO formation in EOC13 microglial cells. Adherent DAF-FM DA-loaded EOC13 cells in NaCl medium were preincubated in the absence (control) or presence of 10 μ M AZ10606120 at 37°C for 15 min and then in the absence (basal) or presence of 1.4 mM ATP for 15 min. Incubations were stopped by the addition of MgCl 2 medium and centrifugation. Mean fluorescence intensities (MFI) of benzotriazole (NO formation) were determined by flow cytometry and results shown as means ± SD, n = 3; *** P < 0.001 compared to corresponding basal; ††† P < 0.001 compared to corresponding ATP.

    Journal: Mediators of Inflammation

    Article Title: P2X7 Receptor Activation Induces Reactive Oxygen Species Formation and Cell Death in Murine EOC13 Microglia

    doi: 10.1155/2013/271813

    Figure Lengend Snippet: P2X7 activation induces NO formation in EOC13 microglial cells. Adherent DAF-FM DA-loaded EOC13 cells in NaCl medium were preincubated in the absence (control) or presence of 10 μ M AZ10606120 at 37°C for 15 min and then in the absence (basal) or presence of 1.4 mM ATP for 15 min. Incubations were stopped by the addition of MgCl 2 medium and centrifugation. Mean fluorescence intensities (MFI) of benzotriazole (NO formation) were determined by flow cytometry and results shown as means ± SD, n = 3; *** P < 0.001 compared to corresponding basal; ††† P < 0.001 compared to corresponding ATP.

    Article Snippet: Rat anti-mouse P2X7 monoclonal antibody (mAb) (clone HANO43) was from Enzo Life Sciences.

    Techniques: Activation Assay, Centrifugation, Fluorescence, Flow Cytometry

    P2X7 activation induces cell death in EOC13 microglial cells. (a) Adherent EOC13 cells in complete DMEM medium were incubated in the absence or presence of varying concentrations of ATP (as indicated) at 37°C for 24 h. (b) Adherent cells in complete DMEM medium were preincubated in the absence or presence of 10 μ M AZ10606120 at 37°C for 15 min and then in the absence or presence of 2 mM ATP for 24 h. (e and f) Adherent cells in complete DMEM medium were incubated in the absence or presence of 40 mM NAC at 37°C for 90 min and incubated in the absence or presence of 2 mM ATP for the final (e) 15–60 min or (f) 45 min (of the 90 min incubation), and then the medium replaced with fresh complete DMEM medium for 24 h. (a, b, and e) Cells were harvested, labelled with Annexin-V-Fluorescein and 7AAD, and the percentage of Annexin-V − /7AAD + , Annexin-V + /7AAD − , and Annexin-V + /7AAD + cells (together representing total cell death) determined by flow cytometry. (f) DIC images of cell morphology were acquired by microscopy. Bars represent 20 μ m. (c) Adherent DCF-loaded cells in complete DMEM medium were incubated in the absence (basal) or presence of varying concentrations of ATP (as indicated) at 37°C for 15 min. (d) Adherent DCF-loaded cells in complete DMEM medium were preincubated in the absence (control) or presence of 10 μ M AZ10606120 at 37°C for 15 min and then in the absence (basal) or presence of 2 mM ATP for 15 min. (c and d) Incubations were stopped by the addition of MgCl 2 medium and centrifugation. Mean fluorescence intensity (MFI) of DCF (ROS formation) was determined by flow cytometry. Results shown as (a) dot plots of one representative set of data demonstrating the quadrant markers and (a–e) means ± SD, n = 3; *** P < 0.001 or * P < 0.05 compared to (a and c) 0 mM ATP, (b and d) corresponding basal, or (e) corresponding 0 min ATP; ††† P < 0.001 compared to corresponding ATP in the absence of (b and d) AZ10606120 or (e) NAC.

    Journal: Mediators of Inflammation

    Article Title: P2X7 Receptor Activation Induces Reactive Oxygen Species Formation and Cell Death in Murine EOC13 Microglia

    doi: 10.1155/2013/271813

    Figure Lengend Snippet: P2X7 activation induces cell death in EOC13 microglial cells. (a) Adherent EOC13 cells in complete DMEM medium were incubated in the absence or presence of varying concentrations of ATP (as indicated) at 37°C for 24 h. (b) Adherent cells in complete DMEM medium were preincubated in the absence or presence of 10 μ M AZ10606120 at 37°C for 15 min and then in the absence or presence of 2 mM ATP for 24 h. (e and f) Adherent cells in complete DMEM medium were incubated in the absence or presence of 40 mM NAC at 37°C for 90 min and incubated in the absence or presence of 2 mM ATP for the final (e) 15–60 min or (f) 45 min (of the 90 min incubation), and then the medium replaced with fresh complete DMEM medium for 24 h. (a, b, and e) Cells were harvested, labelled with Annexin-V-Fluorescein and 7AAD, and the percentage of Annexin-V − /7AAD + , Annexin-V + /7AAD − , and Annexin-V + /7AAD + cells (together representing total cell death) determined by flow cytometry. (f) DIC images of cell morphology were acquired by microscopy. Bars represent 20 μ m. (c) Adherent DCF-loaded cells in complete DMEM medium were incubated in the absence (basal) or presence of varying concentrations of ATP (as indicated) at 37°C for 15 min. (d) Adherent DCF-loaded cells in complete DMEM medium were preincubated in the absence (control) or presence of 10 μ M AZ10606120 at 37°C for 15 min and then in the absence (basal) or presence of 2 mM ATP for 15 min. (c and d) Incubations were stopped by the addition of MgCl 2 medium and centrifugation. Mean fluorescence intensity (MFI) of DCF (ROS formation) was determined by flow cytometry. Results shown as (a) dot plots of one representative set of data demonstrating the quadrant markers and (a–e) means ± SD, n = 3; *** P < 0.001 or * P < 0.05 compared to (a and c) 0 mM ATP, (b and d) corresponding basal, or (e) corresponding 0 min ATP; ††† P < 0.001 compared to corresponding ATP in the absence of (b and d) AZ10606120 or (e) NAC.

    Article Snippet: Rat anti-mouse P2X7 monoclonal antibody (mAb) (clone HANO43) was from Enzo Life Sciences.

    Techniques: Activation Assay, Incubation, Flow Cytometry, Microscopy, Centrifugation, Fluorescence

    Complex formation between Xk and Vps13a in WR19L cells. ( A ) BN-PAGE analysis of Xk and Vps13a in WR19L cells. The solubilized crude membrane fractions (4.2 µg of protein) and the cytosolic fractions (21 µg of protein) from DKO , DKO -P2X7, Xk −/− DKO -P2X7 ( Xk −/− ), and Vps13a −/− DKO -P2X7 ( Vps13a −/− ) were separated by BN-PAGE or SDS-PAGE and analyzed by Western blotting with anti-Xk ( Left ) or anti-Vps13a Ab ( Right ). Each membrane was stained with CBB and shown in the Lower panels. The positions of Mr. standard proteins are shown with their Mr. ( B ) The cellular localization of Xk. Xk −/− DKO -P2X7 ( Xk −/− ) and Xk −/− Vps13a −/− DKO -P2X7 ( Xk −/− Vps13a −/− ) cells were transformed with Xk–EGFP and observed by confocal microscopy in the presence of 5 µg/mL Hoechst 33342. EGFP and Hoechst signals are shown in green and blue. (Scale bars, 10 µm.)

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Requirement of Xk and Vps13a for the P2X7-mediated phospholipid scrambling and cell lysis in mouse T cells

    doi: 10.1073/pnas.2119286119

    Figure Lengend Snippet: Complex formation between Xk and Vps13a in WR19L cells. ( A ) BN-PAGE analysis of Xk and Vps13a in WR19L cells. The solubilized crude membrane fractions (4.2 µg of protein) and the cytosolic fractions (21 µg of protein) from DKO , DKO -P2X7, Xk −/− DKO -P2X7 ( Xk −/− ), and Vps13a −/− DKO -P2X7 ( Vps13a −/− ) were separated by BN-PAGE or SDS-PAGE and analyzed by Western blotting with anti-Xk ( Left ) or anti-Vps13a Ab ( Right ). Each membrane was stained with CBB and shown in the Lower panels. The positions of Mr. standard proteins are shown with their Mr. ( B ) The cellular localization of Xk. Xk −/− DKO -P2X7 ( Xk −/− ) and Xk −/− Vps13a −/− DKO -P2X7 ( Xk −/− Vps13a −/− ) cells were transformed with Xk–EGFP and observed by confocal microscopy in the presence of 5 µg/mL Hoechst 33342. EGFP and Hoechst signals are shown in green and blue. (Scale bars, 10 µm.)

    Article Snippet: Alexa 647-conjugated rat anti-mouse P2X7 monoclonal Ab (mAb) (clone Hano43) was from Bio-Rad Laboratories.

    Techniques: SDS Page, Western Blot, Staining, Transformation Assay, Confocal Microscopy