rabbit anti rat p2x7  (Alomone Labs)


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    Alomone Labs rabbit anti rat p2x7
    <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.
    Rabbit Anti Rat P2x7, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit anti rat p2x7/product/Alomone Labs
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    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

    affinity purified polyclonal rabbit anti rat p2x 7 r  (Alomone Labs)


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    Alomone Labs affinity purified polyclonal rabbit anti rat p2x 7 r
    Confocal fluorescence Z stack images of flat mount bladder mucosa taken from the urothelial towards the serosal surface. (A) Granular staining for Panx1 channels (red) is observed throughout the mucosa. Staining for the intermediate filament vimentin (green) is observed on the apical urothelial region and particularly on a few cells in the lamina propria, which likely correspond to suburothelial myofibroblasts. Note partial colocalization of Pannexin 1 with vimentin-positive cells. (B) Positive staining for <t>P2X</t> <t>7</t> <t>R</t> is observed in the urothelium, blood vessels (white arrows) and lamina propria, while staining for cytokeratin 7/17 is restricted to urothelial cells. Note intense P2X 7 R immunoreactivity on the basal region of the mucosa, which is likely localized to the lamina propria myofibroblasts. DAPI nuclear staining in blue. Scale bar = 20 µm.
    Affinity Purified Polyclonal Rabbit Anti Rat P2x 7 R, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Pannexin 1 Channels Play Essential Roles in Urothelial Mechanotransduction and Intercellular Signaling"

    Article Title: Pannexin 1 Channels Play Essential Roles in Urothelial Mechanotransduction and Intercellular Signaling

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0106269

    Confocal fluorescence Z stack images of flat mount bladder mucosa taken from the urothelial towards the serosal surface. (A) Granular staining for Panx1 channels (red) is observed throughout the mucosa. Staining for the intermediate filament vimentin (green) is observed on the apical urothelial region and particularly on a few cells in the lamina propria, which likely correspond to suburothelial myofibroblasts. Note partial colocalization of Pannexin 1 with vimentin-positive cells. (B) Positive staining for P2X 7 R is observed in the urothelium, blood vessels (white arrows) and lamina propria, while staining for cytokeratin 7/17 is restricted to urothelial cells. Note intense P2X 7 R immunoreactivity on the basal region of the mucosa, which is likely localized to the lamina propria myofibroblasts. DAPI nuclear staining in blue. Scale bar = 20 µm.
    Figure Legend Snippet: Confocal fluorescence Z stack images of flat mount bladder mucosa taken from the urothelial towards the serosal surface. (A) Granular staining for Panx1 channels (red) is observed throughout the mucosa. Staining for the intermediate filament vimentin (green) is observed on the apical urothelial region and particularly on a few cells in the lamina propria, which likely correspond to suburothelial myofibroblasts. Note partial colocalization of Pannexin 1 with vimentin-positive cells. (B) Positive staining for P2X 7 R is observed in the urothelium, blood vessels (white arrows) and lamina propria, while staining for cytokeratin 7/17 is restricted to urothelial cells. Note intense P2X 7 R immunoreactivity on the basal region of the mucosa, which is likely localized to the lamina propria myofibroblasts. DAPI nuclear staining in blue. Scale bar = 20 µm.

    Techniques Used: Fluorescence, Staining

    Whole bladders isolated from wildtype (WT), Panx1 deficient (Panx1 −/− ) and P2X 7 R deficient (P2X 7 R −/− ) mice were bathed and instilled with PBS+glucose (1 g/L). A filling-voiding cycle was simulated by bladder instillation for 8 min at 1.5 mL/h flow rate followed by 5 min no flow, after which the bladder was emptied and ATP release in the bladder lumen was quantified. Data represent mean ± SEM (N = 9 WT, 4 Panx1 −/− and 7 P2X 7 R −/− bladders. Compared to WT: * P< 0.05 and ** P< 0.01 by Student’s t -test).
    Figure Legend Snippet: Whole bladders isolated from wildtype (WT), Panx1 deficient (Panx1 −/− ) and P2X 7 R deficient (P2X 7 R −/− ) mice were bathed and instilled with PBS+glucose (1 g/L). A filling-voiding cycle was simulated by bladder instillation for 8 min at 1.5 mL/h flow rate followed by 5 min no flow, after which the bladder was emptied and ATP release in the bladder lumen was quantified. Data represent mean ± SEM (N = 9 WT, 4 Panx1 −/− and 7 P2X 7 R −/− bladders. Compared to WT: * P< 0.05 and ** P< 0.01 by Student’s t -test).

    Techniques Used: Isolation

    Detection of Panx1 and P2X 7 R mRNA by PCR in (A) and protein by immunoblotting in (B). Total RNA from human bladder and HeLa cells were used as reference for the PCR analyses, and whole HeLa cell lysates was used as reference for immunoblotting.
    Figure Legend Snippet: Detection of Panx1 and P2X 7 R mRNA by PCR in (A) and protein by immunoblotting in (B). Total RNA from human bladder and HeLa cells were used as reference for the PCR analyses, and whole HeLa cell lysates was used as reference for immunoblotting.

    Techniques Used: Western Blot

    (A) TRT-HU1 cells: YoPro-1 uptake induced by cell swelling (hypoosmotic shock, black line) was significantly reduced in the presence of the Panx1 channel blocker mefloquine (MFQ 100 nM; yellow line), the P2X 7 R blocker A438079 (10 µM; red line) and when both Panx1 and P2X 7 R were blocked (blue line). Except for hypoosmotic with MFQ vs. A438079 and hypoosmotic with A438079+MFQ vs. isoosmotic, all other comparisons were significantly different at time 2800 sec ( P <0.01, N≥6) by two-way repeated measures ANOVA, followed by Tukey’s multiple comparison. (B) Primary mouse urothelial cells: YoPro-1 uptake induced by hypoosmotic shock was significantly lower in P2X 7 R −/− (red line) compared to wildtype (WT) urothelial cells (black line). Dye uptake by P2X 7 R −/− cells was abolished in the presence of the Panx1 channel blocker mefloquine (MFQ, 100 nM; green line) and was absent in Panx1 −/− urothelial cells (blue line). WT vs P2X 7 R −/− , P2X 7 R −/− +MFQ and Panx1 −/− ( P< 0.001, N = 4) by ANOVA followed by Tukey’s multiple comparison.
    Figure Legend Snippet: (A) TRT-HU1 cells: YoPro-1 uptake induced by cell swelling (hypoosmotic shock, black line) was significantly reduced in the presence of the Panx1 channel blocker mefloquine (MFQ 100 nM; yellow line), the P2X 7 R blocker A438079 (10 µM; red line) and when both Panx1 and P2X 7 R were blocked (blue line). Except for hypoosmotic with MFQ vs. A438079 and hypoosmotic with A438079+MFQ vs. isoosmotic, all other comparisons were significantly different at time 2800 sec ( P <0.01, N≥6) by two-way repeated measures ANOVA, followed by Tukey’s multiple comparison. (B) Primary mouse urothelial cells: YoPro-1 uptake induced by hypoosmotic shock was significantly lower in P2X 7 R −/− (red line) compared to wildtype (WT) urothelial cells (black line). Dye uptake by P2X 7 R −/− cells was abolished in the presence of the Panx1 channel blocker mefloquine (MFQ, 100 nM; green line) and was absent in Panx1 −/− urothelial cells (blue line). WT vs P2X 7 R −/− , P2X 7 R −/− +MFQ and Panx1 −/− ( P< 0.001, N = 4) by ANOVA followed by Tukey’s multiple comparison.

    Techniques Used:

    (A) Mechanical stimulation imposed by rinsing the cells with bathing solution induced ATP release from TRT-HU1 cells that was significantly higher than that from non-stimulated cells when measured in the presence or absence of the Panx1 channel blocker mefloquine (MFQ, 100 nM; n = 4 each, * P <0.05 and ** P <0.01, by paired t -test.). (B) Normalized ATP release with respect to basal values, however, was significantly lower in MFQ-treated compared to untreated cells (N = 4, $ P <0.05 by Mann-Whitney U test). (C) Exposure to low divalent cation solution (LDPBS), a condition known to enhance P2X 7 R activation, significantly increased ATP release from TRT-HU1 cells. All data represent mean ± SEM (N = 4 each, * P <0.05 by Student’s t -test).
    Figure Legend Snippet: (A) Mechanical stimulation imposed by rinsing the cells with bathing solution induced ATP release from TRT-HU1 cells that was significantly higher than that from non-stimulated cells when measured in the presence or absence of the Panx1 channel blocker mefloquine (MFQ, 100 nM; n = 4 each, * P <0.05 and ** P <0.01, by paired t -test.). (B) Normalized ATP release with respect to basal values, however, was significantly lower in MFQ-treated compared to untreated cells (N = 4, $ P <0.05 by Mann-Whitney U test). (C) Exposure to low divalent cation solution (LDPBS), a condition known to enhance P2X 7 R activation, significantly increased ATP release from TRT-HU1 cells. All data represent mean ± SEM (N = 4 each, * P <0.05 by Student’s t -test).

    Techniques Used: MANN-WHITNEY, Activation Assay

    rat antipurinergic receptor p2x ligand gated ion channel 7  (Santa Cruz Biotechnology)

     
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    Santa Cruz Biotechnology rat antipurinergic receptor p2x ligand gated ion channel 7
    Ruscogenin promoted the AQP5 and AQP4 expression while suppressed inflammation-related factors expression in submandibular gland tissues of NOD/ShiLtJ mice. (a and b) Representative images of protein bands (a) and relative protein expression of AQP5 and AQP4 (b) in submandibular gland tissues was tested by western blot after treatment of vehicle and Ruscogenin. GAPDH is a loading control. (c and d) Representative images of protein bands (c) and relative protein expression of <t>P2X7R,</t> NLRP3, caspase 1, and IL-1 β (d) in submandibular gland tissues was assessed by western blot after treatment of vehicle and Ruscogenin. GAPDH is a loading control. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001 vs. vehicle group. All experiments were repeated independently at least three times. Data were performed as the means ± standard deviation. AQP: aquaporin; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; P2X7R: purinergic receptor <t>P2X</t> ligand-gated ion channel 7; NLRP3: nucleotide binding oligomerization domain-like receptor 3; IL: interleukin.
    Rat Antipurinergic Receptor P2x Ligand Gated Ion Channel 7, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Ruscogenin Ameliorated Sjögren's Syndrome by Inhibiting NLRP3 Inflammasome Activation"

    Article Title: Ruscogenin Ameliorated Sjögren's Syndrome by Inhibiting NLRP3 Inflammasome Activation

    Journal: Evidence-based Complementary and Alternative Medicine : eCAM

    doi: 10.1155/2022/6425121

    Ruscogenin promoted the AQP5 and AQP4 expression while suppressed inflammation-related factors expression in submandibular gland tissues of NOD/ShiLtJ mice. (a and b) Representative images of protein bands (a) and relative protein expression of AQP5 and AQP4 (b) in submandibular gland tissues was tested by western blot after treatment of vehicle and Ruscogenin. GAPDH is a loading control. (c and d) Representative images of protein bands (c) and relative protein expression of P2X7R, NLRP3, caspase 1, and IL-1 β (d) in submandibular gland tissues was assessed by western blot after treatment of vehicle and Ruscogenin. GAPDH is a loading control. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001 vs. vehicle group. All experiments were repeated independently at least three times. Data were performed as the means ± standard deviation. AQP: aquaporin; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; P2X7R: purinergic receptor P2X ligand-gated ion channel 7; NLRP3: nucleotide binding oligomerization domain-like receptor 3; IL: interleukin.
    Figure Legend Snippet: Ruscogenin promoted the AQP5 and AQP4 expression while suppressed inflammation-related factors expression in submandibular gland tissues of NOD/ShiLtJ mice. (a and b) Representative images of protein bands (a) and relative protein expression of AQP5 and AQP4 (b) in submandibular gland tissues was tested by western blot after treatment of vehicle and Ruscogenin. GAPDH is a loading control. (c and d) Representative images of protein bands (c) and relative protein expression of P2X7R, NLRP3, caspase 1, and IL-1 β (d) in submandibular gland tissues was assessed by western blot after treatment of vehicle and Ruscogenin. GAPDH is a loading control. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001 vs. vehicle group. All experiments were repeated independently at least three times. Data were performed as the means ± standard deviation. AQP: aquaporin; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; P2X7R: purinergic receptor P2X ligand-gated ion channel 7; NLRP3: nucleotide binding oligomerization domain-like receptor 3; IL: interleukin.

    Techniques Used: Expressing, Western Blot, Standard Deviation, Binding Assay

    rabbit anti rat p2x 7 receptor antibody  (Cell Signaling Technology Inc)


    Bioz Manufacturer Symbol Cell Signaling Technology Inc manufactures this product  
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    Cell Signaling Technology Inc rabbit anti rat p2x 7 receptor antibody
    Rabbit Anti Rat P2x 7 Receptor Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    goat anti rat p2x 7 receptors antibody  (Abcam)

     
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    Abcam goat anti rat p2x 7 receptors antibody
    Activation of hippocampal PRG-1 and P2X7 receptor in BCP rats. (A) DAPI overview of a rat showing areas of CA1 (highlighted by white square). (B) Images of PRG-1 (red) and <t>P2X</t> <t>7</t> receptor (green) in the hippocampus from sham and BCP rats by immunofluorescence on the POD 45 (scale bar = 50 μm). (C) Band of western blot and (D-E) quantitative analysis showed that the expression levels of PRG-1 and P2X 7 receptor were increased in the hippocampus of BCP rats compared with corresponding sham group (n = 6, one sample t test). Values represent mean ± SEM. *** P < 0.001. (F-G) Quantitative analysis of qRT-PCR showed that the transcriptions of PRG-1 and P2X 7 receptor mRNA were not increased in the hippocampus of BCP rats. β-actin was included as a control. The data are expressed as the mean ± SEM (n = 6). F: Mann-Whithnes-U test, G: unpaired Student t test, ** P <0.01, *** P <0.001 compared to the sham group. BCP, bone cancer pain.
    Goat Anti Rat P2x 7 Receptors Antibody, supplied by Abcam, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "PRG-1 relieves pain and depressive-like behaviors in rats of bone cancer pain by regulation of dendritic spine in hippocampus"

    Article Title: PRG-1 relieves pain and depressive-like behaviors in rats of bone cancer pain by regulation of dendritic spine in hippocampus

    Journal: International Journal of Biological Sciences

    doi: 10.7150/ijbs.59032

    Activation of hippocampal PRG-1 and P2X7 receptor in BCP rats. (A) DAPI overview of a rat showing areas of CA1 (highlighted by white square). (B) Images of PRG-1 (red) and P2X 7 receptor (green) in the hippocampus from sham and BCP rats by immunofluorescence on the POD 45 (scale bar = 50 μm). (C) Band of western blot and (D-E) quantitative analysis showed that the expression levels of PRG-1 and P2X 7 receptor were increased in the hippocampus of BCP rats compared with corresponding sham group (n = 6, one sample t test). Values represent mean ± SEM. *** P < 0.001. (F-G) Quantitative analysis of qRT-PCR showed that the transcriptions of PRG-1 and P2X 7 receptor mRNA were not increased in the hippocampus of BCP rats. β-actin was included as a control. The data are expressed as the mean ± SEM (n = 6). F: Mann-Whithnes-U test, G: unpaired Student t test, ** P <0.01, *** P <0.001 compared to the sham group. BCP, bone cancer pain.
    Figure Legend Snippet: Activation of hippocampal PRG-1 and P2X7 receptor in BCP rats. (A) DAPI overview of a rat showing areas of CA1 (highlighted by white square). (B) Images of PRG-1 (red) and P2X 7 receptor (green) in the hippocampus from sham and BCP rats by immunofluorescence on the POD 45 (scale bar = 50 μm). (C) Band of western blot and (D-E) quantitative analysis showed that the expression levels of PRG-1 and P2X 7 receptor were increased in the hippocampus of BCP rats compared with corresponding sham group (n = 6, one sample t test). Values represent mean ± SEM. *** P < 0.001. (F-G) Quantitative analysis of qRT-PCR showed that the transcriptions of PRG-1 and P2X 7 receptor mRNA were not increased in the hippocampus of BCP rats. β-actin was included as a control. The data are expressed as the mean ± SEM (n = 6). F: Mann-Whithnes-U test, G: unpaired Student t test, ** P <0.01, *** P <0.001 compared to the sham group. BCP, bone cancer pain.

    Techniques Used: Activation Assay, Immunofluorescence, Western Blot, Expressing, Quantitative RT-PCR

    PRG-1 attenuated pain and depression-like behaviors in BCP rats. (A) Schematic diagram of hippocampal microinjection (x = ±2.0 mm, y = -3.72 mm, and z = -3.0 mm). (B) Hippocampus injection of virus vector LV-Plppr4 (PRG-1 OE) and LV-P2X7R-RNAi (P2X 7 receptor KD) alleviated BCP-induced TWL decreases while LV-Plppr4-RNAi (PRG-1 KD) lead to early and intensified pain (n = 12; one-way ANOVA, *: versus Sham group at the same time point; #: versus BCP group at the same time point). (C) Hippocampus injection of virus vector LV-Plppr4 (PRG-1 OE) and LV-P2X7R-RNAi (P2X 7 receptor KD) alleviated BCP-induced MWT decreases while LV-Plppr4-RNAi (PRG-1 KD) lead to early and intensified pain (n = 12; one-way ANOVA, *: versus Sham group at the same time point; #: versus BCP group at the same time point). (D) PRG-1 OE reversed the preference of sucrose (n = 12; one-way ANOVA, #: versus BCP group at the same time point). (E) Multiple FTY720 administration attenuated BCP-induced thermal withdrawal latency decreases from day 39 to 43 (n = 12; one-way ANOVA, *: versus Sham group at the same time point; #: versus BCP group at the same time point); dotted lines indicate pharmacological treatment). (F) Enlarged graph of D from POD 38 to 44. (G) Chronic FTY720 administration attenuated BCP-induced MWT decreases on day 40. (n = 6; Kruskal-Wallis test. #: versus BCP group at the same time point); dotted lines indicate pharmacological treatment). (H) FTY720 reversed the decline in the preference of sucrose consumption induced by BCP (n = 12 one-way ANOVA, *: versus Sham group at the same time point; #: versus BCP group at the same time point). The data are presented mean ± SEM. * P <0.05, ** P <0.01, *** P <0.001; BCP, bone cancer pain; TWL, thermal withdrawal latency; MWT, mechanical withdrawal threshold; OE, overexpression; KD, knock down.
    Figure Legend Snippet: PRG-1 attenuated pain and depression-like behaviors in BCP rats. (A) Schematic diagram of hippocampal microinjection (x = ±2.0 mm, y = -3.72 mm, and z = -3.0 mm). (B) Hippocampus injection of virus vector LV-Plppr4 (PRG-1 OE) and LV-P2X7R-RNAi (P2X 7 receptor KD) alleviated BCP-induced TWL decreases while LV-Plppr4-RNAi (PRG-1 KD) lead to early and intensified pain (n = 12; one-way ANOVA, *: versus Sham group at the same time point; #: versus BCP group at the same time point). (C) Hippocampus injection of virus vector LV-Plppr4 (PRG-1 OE) and LV-P2X7R-RNAi (P2X 7 receptor KD) alleviated BCP-induced MWT decreases while LV-Plppr4-RNAi (PRG-1 KD) lead to early and intensified pain (n = 12; one-way ANOVA, *: versus Sham group at the same time point; #: versus BCP group at the same time point). (D) PRG-1 OE reversed the preference of sucrose (n = 12; one-way ANOVA, #: versus BCP group at the same time point). (E) Multiple FTY720 administration attenuated BCP-induced thermal withdrawal latency decreases from day 39 to 43 (n = 12; one-way ANOVA, *: versus Sham group at the same time point; #: versus BCP group at the same time point); dotted lines indicate pharmacological treatment). (F) Enlarged graph of D from POD 38 to 44. (G) Chronic FTY720 administration attenuated BCP-induced MWT decreases on day 40. (n = 6; Kruskal-Wallis test. #: versus BCP group at the same time point); dotted lines indicate pharmacological treatment). (H) FTY720 reversed the decline in the preference of sucrose consumption induced by BCP (n = 12 one-way ANOVA, *: versus Sham group at the same time point; #: versus BCP group at the same time point). The data are presented mean ± SEM. * P <0.05, ** P <0.01, *** P <0.001; BCP, bone cancer pain; TWL, thermal withdrawal latency; MWT, mechanical withdrawal threshold; OE, overexpression; KD, knock down.

    Techniques Used: Injection, Plasmid Preparation, Over Expression

    PRG-1 attenuated neuron deactivation and synaptic depression in the hippocampus induced by BCP. (A) Nissl staining in CA1, DG districts of rats' hippocampus under FTY720 or A438079 treatment. (B) The graph shows the percent of nissl positive cell. n = 6; one-way ANOVA, *: versus Sham group. (C) Images of Golgi-Cox staining and (D-F) quantitative analysis showed that the spine density depression induced by BCP was reversed by FTY720. (D, n =20 Sham, 12 BCP, 14 BCP+FTY720 and 13 BCP+A438079, one-way ANOVA; E, n = 20 Sham,11 BCP, 12 BCP+FTY720 and 12 BCP+A438079, one-way ANOVA; F, n = 12 Sham, 10 BCP, 15 BCP+FTY720 and 15 BCP+A438079, Kruskal-Wallis test; n represents analyzed dendritic segments; *: versus Sham group; #: versus BCP group); stratum radiatum (sr, apical dendrites) and stratum oriens (so, basal dendrites) of the CA1 region, and the stratum moleculare of the dentate gyrus (moDG). (G) PRG-1 OE and P2X 7 R KD rescued nissl bodies and nucleoli in CA1 and DG regions. The data are expressed as the mean ± SEM; * P <0.05, ** P <0.01, *** P <0.001, BCP, bone cancer pain; OE, overexpression; KD, knock down.
    Figure Legend Snippet: PRG-1 attenuated neuron deactivation and synaptic depression in the hippocampus induced by BCP. (A) Nissl staining in CA1, DG districts of rats' hippocampus under FTY720 or A438079 treatment. (B) The graph shows the percent of nissl positive cell. n = 6; one-way ANOVA, *: versus Sham group. (C) Images of Golgi-Cox staining and (D-F) quantitative analysis showed that the spine density depression induced by BCP was reversed by FTY720. (D, n =20 Sham, 12 BCP, 14 BCP+FTY720 and 13 BCP+A438079, one-way ANOVA; E, n = 20 Sham,11 BCP, 12 BCP+FTY720 and 12 BCP+A438079, one-way ANOVA; F, n = 12 Sham, 10 BCP, 15 BCP+FTY720 and 15 BCP+A438079, Kruskal-Wallis test; n represents analyzed dendritic segments; *: versus Sham group; #: versus BCP group); stratum radiatum (sr, apical dendrites) and stratum oriens (so, basal dendrites) of the CA1 region, and the stratum moleculare of the dentate gyrus (moDG). (G) PRG-1 OE and P2X 7 R KD rescued nissl bodies and nucleoli in CA1 and DG regions. The data are expressed as the mean ± SEM; * P <0.05, ** P <0.01, *** P <0.001, BCP, bone cancer pain; OE, overexpression; KD, knock down.

    Techniques Used: Staining, Over Expression

    PRG-1/PP2A interaction were increased by hippocampal injections of FTY720. (A) Images of PRG-1 (red) and P2X 7 receptor (green) in the hippocampus from BCP, BCP+FTY720 and BCP+A438079 rats by immunofluorescence on the POD 44 (scale bar = 50 μm). (B) Western blot showed that the expression of PRG-1 and P2X 7 receptor was increased in the hippocampus of BCP+FTY720 rats. (C) Co-immunoprecipitation (IP) using a PRG-1 antibody shows PRG-1 and PP2A association. (D-E) Quantitative analysis of RT-qPCR showed that the transcription of PRG-1 and P2X7 receptor mRNA was increased in the hippocampus of BCP+FTY720 rats. β-actin was included as a control. The data are expressed as the mean ± SEM; n = 6; D: Kruskal-Wallis test, E: one-way ANOVA; * P <0.05, ** P <0.005, *** P <0.001 compared to the BCP group. (F) Band of western blot showed the expression of PRG-1 and P2X 7 receptor induced by virus expression vectors. BCP, bone cancer pain; OE, overexpression; KD, knock down; POD, postoperative day.
    Figure Legend Snippet: PRG-1/PP2A interaction were increased by hippocampal injections of FTY720. (A) Images of PRG-1 (red) and P2X 7 receptor (green) in the hippocampus from BCP, BCP+FTY720 and BCP+A438079 rats by immunofluorescence on the POD 44 (scale bar = 50 μm). (B) Western blot showed that the expression of PRG-1 and P2X 7 receptor was increased in the hippocampus of BCP+FTY720 rats. (C) Co-immunoprecipitation (IP) using a PRG-1 antibody shows PRG-1 and PP2A association. (D-E) Quantitative analysis of RT-qPCR showed that the transcription of PRG-1 and P2X7 receptor mRNA was increased in the hippocampus of BCP+FTY720 rats. β-actin was included as a control. The data are expressed as the mean ± SEM; n = 6; D: Kruskal-Wallis test, E: one-way ANOVA; * P <0.05, ** P <0.005, *** P <0.001 compared to the BCP group. (F) Band of western blot showed the expression of PRG-1 and P2X 7 receptor induced by virus expression vectors. BCP, bone cancer pain; OE, overexpression; KD, knock down; POD, postoperative day.

    Techniques Used: Immunofluorescence, Western Blot, Expressing, Immunoprecipitation, Quantitative RT-PCR, Over Expression

    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
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    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

    rabbit anti rat p2x7  (Alomone Labs)


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    Alomone Labs rabbit anti rat p2x7
    <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.
    Rabbit Anti Rat P2x7, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit anti rat p2x7/product/Alomone Labs
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    94/100 stars

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    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

    rabbit polyclonal antibody against rat p2x7  (Abcam)

     
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    Abcam rabbit polyclonal antibody against rat p2x7
    Immunohistochemical staining of the dental pulp of rat. Normal group: A1 crown pulp; A2 upper 1/3 of the root; A3 middle 1/3 of the root; and A4 root tip 1/3; NS group: B1 crown pulp; B2 upper 1/3 of the root; B3 middle 1/3 of the root; and B4 root tip 1/3; LPS group: C1 crown pulp; C2 upper 1/3 of the root; C3 middle 1/3 of the root; and C4 root tip 1/3; Transverse images of the mesial root were selected for the pulp parts, the arrows indicate the <t>P2X7</t> receptor expressed in the odontoblast layer in a yellowish-brown granular form. Scale bar = 50 μm.
    Rabbit Polyclonal Antibody Against Rat P2x7, supplied by Abcam, 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/rabbit polyclonal antibody against rat p2x7/product/Abcam
    Average 86 stars, based on 1 article reviews
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    Images

    1) Product Images from "Activation of the P2X7 receptor in the dental pulp tissue contributes to the pain in rats with acute pulpitis"

    Article Title: Activation of the P2X7 receptor in the dental pulp tissue contributes to the pain in rats with acute pulpitis

    Journal: Molecular Pain

    doi: 10.1177/17448069221106844

    Immunohistochemical staining of the dental pulp of rat. Normal group: A1 crown pulp; A2 upper 1/3 of the root; A3 middle 1/3 of the root; and A4 root tip 1/3; NS group: B1 crown pulp; B2 upper 1/3 of the root; B3 middle 1/3 of the root; and B4 root tip 1/3; LPS group: C1 crown pulp; C2 upper 1/3 of the root; C3 middle 1/3 of the root; and C4 root tip 1/3; Transverse images of the mesial root were selected for the pulp parts, the arrows indicate the P2X7 receptor expressed in the odontoblast layer in a yellowish-brown granular form. Scale bar = 50 μm.
    Figure Legend Snippet: Immunohistochemical staining of the dental pulp of rat. Normal group: A1 crown pulp; A2 upper 1/3 of the root; A3 middle 1/3 of the root; and A4 root tip 1/3; NS group: B1 crown pulp; B2 upper 1/3 of the root; B3 middle 1/3 of the root; and B4 root tip 1/3; LPS group: C1 crown pulp; C2 upper 1/3 of the root; C3 middle 1/3 of the root; and C4 root tip 1/3; Transverse images of the mesial root were selected for the pulp parts, the arrows indicate the P2X7 receptor expressed in the odontoblast layer in a yellowish-brown granular form. Scale bar = 50 μm.

    Techniques Used: Immunohistochemical staining, Staining

    Western Blotting bands of the P2X7 receptor protein in the dental pulp tissue of rats in each group.
    Figure Legend Snippet: Western Blotting bands of the P2X7 receptor protein in the dental pulp tissue of rats in each group.

    Techniques Used: Western Blot

    Comparison of the P2X7 receptor protein expression in the dental pulp tissue of rats in each group. *** p < 0.001, compared to the normal group; and ▲▲▲ p < 0.001, compared to the NS group.
    Figure Legend Snippet: Comparison of the P2X7 receptor protein expression in the dental pulp tissue of rats in each group. *** p < 0.001, compared to the normal group; and ▲▲▲ p < 0.001, compared to the NS group.

    Techniques Used: Expressing

    rabbit anti rat p2x7 antibody  (Alomone Labs)


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    Alomone Labs rabbit anti rat p2x7 antibody
    Rabbit Anti Rat P2x7 Antibody, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    rabbit anti rat p2x7 antibody  (Alomone Labs)


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    Alomone Labs rabbit anti rat p2x7 antibody
    Rabbit Anti Rat P2x7 Antibody, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit anti rat p2x7 antibody/product/Alomone Labs
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    alexa 647 conjugated rat anti mouse p2x7 monoclonal ab mab  (Bio-Rad)

     
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    Bio-Rad alexa 647 conjugated rat anti mouse p2x7 monoclonal ab mab
    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 <t>-P2X7,</t> 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.)
    Alexa 647 Conjugated Rat Anti Mouse P2x7 Monoclonal Ab Mab, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/alexa 647 conjugated rat anti mouse p2x7 monoclonal ab mab/product/Bio-Rad
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    Images

    1) Product Images from "Requirement of Xk and Vps13a for the P2X7-mediated phospholipid scrambling and cell lysis in mouse T cells"

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

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

    doi: 10.1073/pnas.2119286119

    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.)
    Figure Legend 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.)

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

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    • rabbit anti rat p2x7  (Alomone Labs)
    94
    Alomone Labs rabbit anti rat p2x7
    <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.
    Rabbit Anti Rat P2x7, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    affinity purified polyclonal rabbit anti rat p2x 7 r  (Alomone Labs)
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    Alomone Labs affinity purified polyclonal rabbit anti rat p2x 7 r
    Confocal fluorescence Z stack images of flat mount bladder mucosa taken from the urothelial towards the serosal surface. (A) Granular staining for Panx1 channels (red) is observed throughout the mucosa. Staining for the intermediate filament vimentin (green) is observed on the apical urothelial region and particularly on a few cells in the lamina propria, which likely correspond to suburothelial myofibroblasts. Note partial colocalization of Pannexin 1 with vimentin-positive cells. (B) Positive staining for <t>P2X</t> <t>7</t> <t>R</t> is observed in the urothelium, blood vessels (white arrows) and lamina propria, while staining for cytokeratin 7/17 is restricted to urothelial cells. Note intense P2X 7 R immunoreactivity on the basal region of the mucosa, which is likely localized to the lamina propria myofibroblasts. DAPI nuclear staining in blue. Scale bar = 20 µm.
    Affinity Purified Polyclonal Rabbit Anti Rat P2x 7 R, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    rat antipurinergic receptor p2x ligand gated ion channel 7  (Santa Cruz Biotechnology)
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    Santa Cruz Biotechnology rat antipurinergic receptor p2x ligand gated ion channel 7
    Ruscogenin promoted the AQP5 and AQP4 expression while suppressed inflammation-related factors expression in submandibular gland tissues of NOD/ShiLtJ mice. (a and b) Representative images of protein bands (a) and relative protein expression of AQP5 and AQP4 (b) in submandibular gland tissues was tested by western blot after treatment of vehicle and Ruscogenin. GAPDH is a loading control. (c and d) Representative images of protein bands (c) and relative protein expression of <t>P2X7R,</t> NLRP3, caspase 1, and IL-1 β (d) in submandibular gland tissues was assessed by western blot after treatment of vehicle and Ruscogenin. GAPDH is a loading control. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001 vs. vehicle group. All experiments were repeated independently at least three times. Data were performed as the means ± standard deviation. AQP: aquaporin; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; P2X7R: purinergic receptor <t>P2X</t> ligand-gated ion channel 7; NLRP3: nucleotide binding oligomerization domain-like receptor 3; IL: interleukin.
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    rabbit anti rat p2x 7 receptor antibody  (Cell Signaling Technology Inc)
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    Cell Signaling Technology Inc rabbit anti rat p2x 7 receptor antibody
    Ruscogenin promoted the AQP5 and AQP4 expression while suppressed inflammation-related factors expression in submandibular gland tissues of NOD/ShiLtJ mice. (a and b) Representative images of protein bands (a) and relative protein expression of AQP5 and AQP4 (b) in submandibular gland tissues was tested by western blot after treatment of vehicle and Ruscogenin. GAPDH is a loading control. (c and d) Representative images of protein bands (c) and relative protein expression of <t>P2X7R,</t> NLRP3, caspase 1, and IL-1 β (d) in submandibular gland tissues was assessed by western blot after treatment of vehicle and Ruscogenin. GAPDH is a loading control. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001 vs. vehicle group. All experiments were repeated independently at least three times. Data were performed as the means ± standard deviation. AQP: aquaporin; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; P2X7R: purinergic receptor <t>P2X</t> ligand-gated ion channel 7; NLRP3: nucleotide binding oligomerization domain-like receptor 3; IL: interleukin.
    Rabbit Anti Rat P2x 7 Receptor Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    goat anti rat p2x 7 receptors antibody  (Abcam)
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    Abcam goat anti rat p2x 7 receptors antibody
    Activation of hippocampal PRG-1 and P2X7 receptor in BCP rats. (A) DAPI overview of a rat showing areas of CA1 (highlighted by white square). (B) Images of PRG-1 (red) and <t>P2X</t> <t>7</t> receptor (green) in the hippocampus from sham and BCP rats by immunofluorescence on the POD 45 (scale bar = 50 μm). (C) Band of western blot and (D-E) quantitative analysis showed that the expression levels of PRG-1 and P2X 7 receptor were increased in the hippocampus of BCP rats compared with corresponding sham group (n = 6, one sample t test). Values represent mean ± SEM. *** P < 0.001. (F-G) Quantitative analysis of qRT-PCR showed that the transcriptions of PRG-1 and P2X 7 receptor mRNA were not increased in the hippocampus of BCP rats. β-actin was included as a control. The data are expressed as the mean ± SEM (n = 6). F: Mann-Whithnes-U test, G: unpaired Student t test, ** P <0.01, *** P <0.001 compared to the sham group. BCP, bone cancer pain.
    Goat Anti Rat P2x 7 Receptors Antibody, supplied by Abcam, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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
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    rabbit polyclonal antibody against rat p2x7  (Abcam)
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    Abcam rabbit polyclonal antibody against rat p2x7
    Immunohistochemical staining of the dental pulp of rat. Normal group: A1 crown pulp; A2 upper 1/3 of the root; A3 middle 1/3 of the root; and A4 root tip 1/3; NS group: B1 crown pulp; B2 upper 1/3 of the root; B3 middle 1/3 of the root; and B4 root tip 1/3; LPS group: C1 crown pulp; C2 upper 1/3 of the root; C3 middle 1/3 of the root; and C4 root tip 1/3; Transverse images of the mesial root were selected for the pulp parts, the arrows indicate the <t>P2X7</t> receptor expressed in the odontoblast layer in a yellowish-brown granular form. Scale bar = 50 μm.
    Rabbit Polyclonal Antibody Against Rat P2x7, supplied by Abcam, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    rabbit anti rat p2x7 antibody  (Alomone Labs)
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    Alomone Labs rabbit anti rat p2x7 antibody
    Immunohistochemical staining of the dental pulp of rat. Normal group: A1 crown pulp; A2 upper 1/3 of the root; A3 middle 1/3 of the root; and A4 root tip 1/3; NS group: B1 crown pulp; B2 upper 1/3 of the root; B3 middle 1/3 of the root; and B4 root tip 1/3; LPS group: C1 crown pulp; C2 upper 1/3 of the root; C3 middle 1/3 of the root; and C4 root tip 1/3; Transverse images of the mesial root were selected for the pulp parts, the arrows indicate the <t>P2X7</t> receptor expressed in the odontoblast layer in a yellowish-brown granular form. Scale bar = 50 μm.
    Rabbit Anti Rat P2x7 Antibody, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit anti rat p2x7 antibody/product/Alomone Labs
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    alexa 647 conjugated rat anti mouse p2x7 monoclonal ab mab  (Bio-Rad)
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    Bio-Rad alexa 647 conjugated rat anti mouse p2x7 monoclonal ab mab
    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 <t>-P2X7,</t> 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.)
    Alexa 647 Conjugated Rat Anti Mouse P2x7 Monoclonal Ab Mab, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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: Rabbit anti-mouse P2X7 (extracellular epitope) polyclonal antibody (Ab) and rabbit anti-rat P2X7 (C-termini epitope) Ab (and corresponding blocking peptide) were from Alomone Labs (Jerusalem, Israel).

    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: Rabbit anti-mouse P2X7 (extracellular epitope) polyclonal antibody (Ab) and rabbit anti-rat P2X7 (C-termini epitope) Ab (and corresponding blocking peptide) were from Alomone Labs (Jerusalem, Israel).

    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: Rabbit anti-mouse P2X7 (extracellular epitope) polyclonal antibody (Ab) and rabbit anti-rat P2X7 (C-termini epitope) Ab (and corresponding blocking peptide) were from Alomone Labs (Jerusalem, Israel).

    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: Rabbit anti-mouse P2X7 (extracellular epitope) polyclonal antibody (Ab) and rabbit anti-rat P2X7 (C-termini epitope) Ab (and corresponding blocking peptide) were from Alomone Labs (Jerusalem, Israel).

    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: Rabbit anti-mouse P2X7 (extracellular epitope) polyclonal antibody (Ab) and rabbit anti-rat P2X7 (C-termini epitope) Ab (and corresponding blocking peptide) were from Alomone Labs (Jerusalem, Israel).

    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: Rabbit anti-mouse P2X7 (extracellular epitope) polyclonal antibody (Ab) and rabbit anti-rat P2X7 (C-termini epitope) Ab (and corresponding blocking peptide) were from Alomone Labs (Jerusalem, Israel).

    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: Rabbit anti-mouse P2X7 (extracellular epitope) polyclonal antibody (Ab) and rabbit anti-rat P2X7 (C-termini epitope) Ab (and corresponding blocking peptide) were from Alomone Labs (Jerusalem, Israel).

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

    Confocal fluorescence Z stack images of flat mount bladder mucosa taken from the urothelial towards the serosal surface. (A) Granular staining for Panx1 channels (red) is observed throughout the mucosa. Staining for the intermediate filament vimentin (green) is observed on the apical urothelial region and particularly on a few cells in the lamina propria, which likely correspond to suburothelial myofibroblasts. Note partial colocalization of Pannexin 1 with vimentin-positive cells. (B) Positive staining for P2X 7 R is observed in the urothelium, blood vessels (white arrows) and lamina propria, while staining for cytokeratin 7/17 is restricted to urothelial cells. Note intense P2X 7 R immunoreactivity on the basal region of the mucosa, which is likely localized to the lamina propria myofibroblasts. DAPI nuclear staining in blue. Scale bar = 20 µm.

    Journal: PLoS ONE

    Article Title: Pannexin 1 Channels Play Essential Roles in Urothelial Mechanotransduction and Intercellular Signaling

    doi: 10.1371/journal.pone.0106269

    Figure Lengend Snippet: Confocal fluorescence Z stack images of flat mount bladder mucosa taken from the urothelial towards the serosal surface. (A) Granular staining for Panx1 channels (red) is observed throughout the mucosa. Staining for the intermediate filament vimentin (green) is observed on the apical urothelial region and particularly on a few cells in the lamina propria, which likely correspond to suburothelial myofibroblasts. Note partial colocalization of Pannexin 1 with vimentin-positive cells. (B) Positive staining for P2X 7 R is observed in the urothelium, blood vessels (white arrows) and lamina propria, while staining for cytokeratin 7/17 is restricted to urothelial cells. Note intense P2X 7 R immunoreactivity on the basal region of the mucosa, which is likely localized to the lamina propria myofibroblasts. DAPI nuclear staining in blue. Scale bar = 20 µm.

    Article Snippet: The following primary antibodies were used: affinity purified polyclonal rabbit anti-rat P2X 7 R corresponding to amino acid residues 576–595 (1∶250, Alomone Labs, Jerusalem, Israel), polyclonal rabbit anti-mouse Pannexin 1 CL (Cytoplasmic loop, 1∶50, Invitrogen, Carlsbad, CA), monoclonal mouse anti-human cytokeratin 7/17 (1∶100, Santa Cruz Biotechnology, Dallas, TX) and monoclonal mouse anti-vimentin (1∶100, Sigma-Aldrich).

    Techniques: Fluorescence, Staining

    Whole bladders isolated from wildtype (WT), Panx1 deficient (Panx1 −/− ) and P2X 7 R deficient (P2X 7 R −/− ) mice were bathed and instilled with PBS+glucose (1 g/L). A filling-voiding cycle was simulated by bladder instillation for 8 min at 1.5 mL/h flow rate followed by 5 min no flow, after which the bladder was emptied and ATP release in the bladder lumen was quantified. Data represent mean ± SEM (N = 9 WT, 4 Panx1 −/− and 7 P2X 7 R −/− bladders. Compared to WT: * P< 0.05 and ** P< 0.01 by Student’s t -test).

    Journal: PLoS ONE

    Article Title: Pannexin 1 Channels Play Essential Roles in Urothelial Mechanotransduction and Intercellular Signaling

    doi: 10.1371/journal.pone.0106269

    Figure Lengend Snippet: Whole bladders isolated from wildtype (WT), Panx1 deficient (Panx1 −/− ) and P2X 7 R deficient (P2X 7 R −/− ) mice were bathed and instilled with PBS+glucose (1 g/L). A filling-voiding cycle was simulated by bladder instillation for 8 min at 1.5 mL/h flow rate followed by 5 min no flow, after which the bladder was emptied and ATP release in the bladder lumen was quantified. Data represent mean ± SEM (N = 9 WT, 4 Panx1 −/− and 7 P2X 7 R −/− bladders. Compared to WT: * P< 0.05 and ** P< 0.01 by Student’s t -test).

    Article Snippet: The following primary antibodies were used: affinity purified polyclonal rabbit anti-rat P2X 7 R corresponding to amino acid residues 576–595 (1∶250, Alomone Labs, Jerusalem, Israel), polyclonal rabbit anti-mouse Pannexin 1 CL (Cytoplasmic loop, 1∶50, Invitrogen, Carlsbad, CA), monoclonal mouse anti-human cytokeratin 7/17 (1∶100, Santa Cruz Biotechnology, Dallas, TX) and monoclonal mouse anti-vimentin (1∶100, Sigma-Aldrich).

    Techniques: Isolation

    Detection of Panx1 and P2X 7 R mRNA by PCR in (A) and protein by immunoblotting in (B). Total RNA from human bladder and HeLa cells were used as reference for the PCR analyses, and whole HeLa cell lysates was used as reference for immunoblotting.

    Journal: PLoS ONE

    Article Title: Pannexin 1 Channels Play Essential Roles in Urothelial Mechanotransduction and Intercellular Signaling

    doi: 10.1371/journal.pone.0106269

    Figure Lengend Snippet: Detection of Panx1 and P2X 7 R mRNA by PCR in (A) and protein by immunoblotting in (B). Total RNA from human bladder and HeLa cells were used as reference for the PCR analyses, and whole HeLa cell lysates was used as reference for immunoblotting.

    Article Snippet: The following primary antibodies were used: affinity purified polyclonal rabbit anti-rat P2X 7 R corresponding to amino acid residues 576–595 (1∶250, Alomone Labs, Jerusalem, Israel), polyclonal rabbit anti-mouse Pannexin 1 CL (Cytoplasmic loop, 1∶50, Invitrogen, Carlsbad, CA), monoclonal mouse anti-human cytokeratin 7/17 (1∶100, Santa Cruz Biotechnology, Dallas, TX) and monoclonal mouse anti-vimentin (1∶100, Sigma-Aldrich).

    Techniques: Western Blot

    (A) TRT-HU1 cells: YoPro-1 uptake induced by cell swelling (hypoosmotic shock, black line) was significantly reduced in the presence of the Panx1 channel blocker mefloquine (MFQ 100 nM; yellow line), the P2X 7 R blocker A438079 (10 µM; red line) and when both Panx1 and P2X 7 R were blocked (blue line). Except for hypoosmotic with MFQ vs. A438079 and hypoosmotic with A438079+MFQ vs. isoosmotic, all other comparisons were significantly different at time 2800 sec ( P <0.01, N≥6) by two-way repeated measures ANOVA, followed by Tukey’s multiple comparison. (B) Primary mouse urothelial cells: YoPro-1 uptake induced by hypoosmotic shock was significantly lower in P2X 7 R −/− (red line) compared to wildtype (WT) urothelial cells (black line). Dye uptake by P2X 7 R −/− cells was abolished in the presence of the Panx1 channel blocker mefloquine (MFQ, 100 nM; green line) and was absent in Panx1 −/− urothelial cells (blue line). WT vs P2X 7 R −/− , P2X 7 R −/− +MFQ and Panx1 −/− ( P< 0.001, N = 4) by ANOVA followed by Tukey’s multiple comparison.

    Journal: PLoS ONE

    Article Title: Pannexin 1 Channels Play Essential Roles in Urothelial Mechanotransduction and Intercellular Signaling

    doi: 10.1371/journal.pone.0106269

    Figure Lengend Snippet: (A) TRT-HU1 cells: YoPro-1 uptake induced by cell swelling (hypoosmotic shock, black line) was significantly reduced in the presence of the Panx1 channel blocker mefloquine (MFQ 100 nM; yellow line), the P2X 7 R blocker A438079 (10 µM; red line) and when both Panx1 and P2X 7 R were blocked (blue line). Except for hypoosmotic with MFQ vs. A438079 and hypoosmotic with A438079+MFQ vs. isoosmotic, all other comparisons were significantly different at time 2800 sec ( P <0.01, N≥6) by two-way repeated measures ANOVA, followed by Tukey’s multiple comparison. (B) Primary mouse urothelial cells: YoPro-1 uptake induced by hypoosmotic shock was significantly lower in P2X 7 R −/− (red line) compared to wildtype (WT) urothelial cells (black line). Dye uptake by P2X 7 R −/− cells was abolished in the presence of the Panx1 channel blocker mefloquine (MFQ, 100 nM; green line) and was absent in Panx1 −/− urothelial cells (blue line). WT vs P2X 7 R −/− , P2X 7 R −/− +MFQ and Panx1 −/− ( P< 0.001, N = 4) by ANOVA followed by Tukey’s multiple comparison.

    Article Snippet: The following primary antibodies were used: affinity purified polyclonal rabbit anti-rat P2X 7 R corresponding to amino acid residues 576–595 (1∶250, Alomone Labs, Jerusalem, Israel), polyclonal rabbit anti-mouse Pannexin 1 CL (Cytoplasmic loop, 1∶50, Invitrogen, Carlsbad, CA), monoclonal mouse anti-human cytokeratin 7/17 (1∶100, Santa Cruz Biotechnology, Dallas, TX) and monoclonal mouse anti-vimentin (1∶100, Sigma-Aldrich).

    Techniques:

    (A) Mechanical stimulation imposed by rinsing the cells with bathing solution induced ATP release from TRT-HU1 cells that was significantly higher than that from non-stimulated cells when measured in the presence or absence of the Panx1 channel blocker mefloquine (MFQ, 100 nM; n = 4 each, * P <0.05 and ** P <0.01, by paired t -test.). (B) Normalized ATP release with respect to basal values, however, was significantly lower in MFQ-treated compared to untreated cells (N = 4, $ P <0.05 by Mann-Whitney U test). (C) Exposure to low divalent cation solution (LDPBS), a condition known to enhance P2X 7 R activation, significantly increased ATP release from TRT-HU1 cells. All data represent mean ± SEM (N = 4 each, * P <0.05 by Student’s t -test).

    Journal: PLoS ONE

    Article Title: Pannexin 1 Channels Play Essential Roles in Urothelial Mechanotransduction and Intercellular Signaling

    doi: 10.1371/journal.pone.0106269

    Figure Lengend Snippet: (A) Mechanical stimulation imposed by rinsing the cells with bathing solution induced ATP release from TRT-HU1 cells that was significantly higher than that from non-stimulated cells when measured in the presence or absence of the Panx1 channel blocker mefloquine (MFQ, 100 nM; n = 4 each, * P <0.05 and ** P <0.01, by paired t -test.). (B) Normalized ATP release with respect to basal values, however, was significantly lower in MFQ-treated compared to untreated cells (N = 4, $ P <0.05 by Mann-Whitney U test). (C) Exposure to low divalent cation solution (LDPBS), a condition known to enhance P2X 7 R activation, significantly increased ATP release from TRT-HU1 cells. All data represent mean ± SEM (N = 4 each, * P <0.05 by Student’s t -test).

    Article Snippet: The following primary antibodies were used: affinity purified polyclonal rabbit anti-rat P2X 7 R corresponding to amino acid residues 576–595 (1∶250, Alomone Labs, Jerusalem, Israel), polyclonal rabbit anti-mouse Pannexin 1 CL (Cytoplasmic loop, 1∶50, Invitrogen, Carlsbad, CA), monoclonal mouse anti-human cytokeratin 7/17 (1∶100, Santa Cruz Biotechnology, Dallas, TX) and monoclonal mouse anti-vimentin (1∶100, Sigma-Aldrich).

    Techniques: MANN-WHITNEY, Activation Assay

    Ruscogenin promoted the AQP5 and AQP4 expression while suppressed inflammation-related factors expression in submandibular gland tissues of NOD/ShiLtJ mice. (a and b) Representative images of protein bands (a) and relative protein expression of AQP5 and AQP4 (b) in submandibular gland tissues was tested by western blot after treatment of vehicle and Ruscogenin. GAPDH is a loading control. (c and d) Representative images of protein bands (c) and relative protein expression of P2X7R, NLRP3, caspase 1, and IL-1 β (d) in submandibular gland tissues was assessed by western blot after treatment of vehicle and Ruscogenin. GAPDH is a loading control. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001 vs. vehicle group. All experiments were repeated independently at least three times. Data were performed as the means ± standard deviation. AQP: aquaporin; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; P2X7R: purinergic receptor P2X ligand-gated ion channel 7; NLRP3: nucleotide binding oligomerization domain-like receptor 3; IL: interleukin.

    Journal: Evidence-based Complementary and Alternative Medicine : eCAM

    Article Title: Ruscogenin Ameliorated Sjögren's Syndrome by Inhibiting NLRP3 Inflammasome Activation

    doi: 10.1155/2022/6425121

    Figure Lengend Snippet: Ruscogenin promoted the AQP5 and AQP4 expression while suppressed inflammation-related factors expression in submandibular gland tissues of NOD/ShiLtJ mice. (a and b) Representative images of protein bands (a) and relative protein expression of AQP5 and AQP4 (b) in submandibular gland tissues was tested by western blot after treatment of vehicle and Ruscogenin. GAPDH is a loading control. (c and d) Representative images of protein bands (c) and relative protein expression of P2X7R, NLRP3, caspase 1, and IL-1 β (d) in submandibular gland tissues was assessed by western blot after treatment of vehicle and Ruscogenin. GAPDH is a loading control. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001 vs. vehicle group. All experiments were repeated independently at least three times. Data were performed as the means ± standard deviation. AQP: aquaporin; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; P2X7R: purinergic receptor P2X ligand-gated ion channel 7; NLRP3: nucleotide binding oligomerization domain-like receptor 3; IL: interleukin.

    Article Snippet: Equal amounts of protein (45 µ g) and ColorMixed Protein Marker (11–180 kDa) (5 µ L; PR1910, Beijing Solarbio Science & Technology Co., Ltd., China) were separated by 6–10% SDS (P0012A, Beyotime Biotechnology, China)-polyacrylamide gel electrophoresis (SDS-PAGE), subsequent to which protein was transferred to PVDF membranes (88585, Thermo Fisher Scientific, USA) blocked in 5% bovine serum albumin (BSA; ST023, Beyotime Biotechnology, China) blocking buffer for 1 h and then incubated at 4°C with rat antipurinergic receptor P2X ligand-gated ion channel 7 (P2X7R, 1 : 1000; sc-134224, Santa Cruz Biotechnology, Dallas, Texas, USA), rabbit anti-NLRP3 (1 : 1000; ab263899, Abcam, Cambridge, MA, USA), rabbit anti-caspase 1 (1 : 1000; ab138483, Abcam, USA), rabbit anti-aquaporin (AQP) 5 (1 : 10000; ab78486, Abcam, USA), rabbit anti-AQP4 (1 : 1000; ab46182, Abcam, USA), rabbit anti-IL-1 β (1 : 1000; #12426, Cell Signaling Technology, Danvers, MA, USA), rabbit anti-Bax (1 : 2000; ab182733, Abcam, USA), Bcl-2 (1 : 2000; ab182858, Abcam, USA), and mouse anti-GAPDH (1 : 10000; ab8245, Abcam, USA) overnight.

    Techniques: Expressing, Western Blot, Standard Deviation, Binding Assay

    Activation of hippocampal PRG-1 and P2X7 receptor in BCP rats. (A) DAPI overview of a rat showing areas of CA1 (highlighted by white square). (B) Images of PRG-1 (red) and P2X 7 receptor (green) in the hippocampus from sham and BCP rats by immunofluorescence on the POD 45 (scale bar = 50 μm). (C) Band of western blot and (D-E) quantitative analysis showed that the expression levels of PRG-1 and P2X 7 receptor were increased in the hippocampus of BCP rats compared with corresponding sham group (n = 6, one sample t test). Values represent mean ± SEM. *** P < 0.001. (F-G) Quantitative analysis of qRT-PCR showed that the transcriptions of PRG-1 and P2X 7 receptor mRNA were not increased in the hippocampus of BCP rats. β-actin was included as a control. The data are expressed as the mean ± SEM (n = 6). F: Mann-Whithnes-U test, G: unpaired Student t test, ** P <0.01, *** P <0.001 compared to the sham group. BCP, bone cancer pain.

    Journal: International Journal of Biological Sciences

    Article Title: PRG-1 relieves pain and depressive-like behaviors in rats of bone cancer pain by regulation of dendritic spine in hippocampus

    doi: 10.7150/ijbs.59032

    Figure Lengend Snippet: Activation of hippocampal PRG-1 and P2X7 receptor in BCP rats. (A) DAPI overview of a rat showing areas of CA1 (highlighted by white square). (B) Images of PRG-1 (red) and P2X 7 receptor (green) in the hippocampus from sham and BCP rats by immunofluorescence on the POD 45 (scale bar = 50 μm). (C) Band of western blot and (D-E) quantitative analysis showed that the expression levels of PRG-1 and P2X 7 receptor were increased in the hippocampus of BCP rats compared with corresponding sham group (n = 6, one sample t test). Values represent mean ± SEM. *** P < 0.001. (F-G) Quantitative analysis of qRT-PCR showed that the transcriptions of PRG-1 and P2X 7 receptor mRNA were not increased in the hippocampus of BCP rats. β-actin was included as a control. The data are expressed as the mean ± SEM (n = 6). F: Mann-Whithnes-U test, G: unpaired Student t test, ** P <0.01, *** P <0.001 compared to the sham group. BCP, bone cancer pain.

    Article Snippet: For immunofluorescence, the sections were blocked and permeabilized, and then incubated with goat anti-rat P2X 7 receptors antibody (1:400, abcam, USA) or rabbit anti-rat PRG-1 antibody (1:500, synaptic system).

    Techniques: Activation Assay, Immunofluorescence, Western Blot, Expressing, Quantitative RT-PCR

    PRG-1 attenuated pain and depression-like behaviors in BCP rats. (A) Schematic diagram of hippocampal microinjection (x = ±2.0 mm, y = -3.72 mm, and z = -3.0 mm). (B) Hippocampus injection of virus vector LV-Plppr4 (PRG-1 OE) and LV-P2X7R-RNAi (P2X 7 receptor KD) alleviated BCP-induced TWL decreases while LV-Plppr4-RNAi (PRG-1 KD) lead to early and intensified pain (n = 12; one-way ANOVA, *: versus Sham group at the same time point; #: versus BCP group at the same time point). (C) Hippocampus injection of virus vector LV-Plppr4 (PRG-1 OE) and LV-P2X7R-RNAi (P2X 7 receptor KD) alleviated BCP-induced MWT decreases while LV-Plppr4-RNAi (PRG-1 KD) lead to early and intensified pain (n = 12; one-way ANOVA, *: versus Sham group at the same time point; #: versus BCP group at the same time point). (D) PRG-1 OE reversed the preference of sucrose (n = 12; one-way ANOVA, #: versus BCP group at the same time point). (E) Multiple FTY720 administration attenuated BCP-induced thermal withdrawal latency decreases from day 39 to 43 (n = 12; one-way ANOVA, *: versus Sham group at the same time point; #: versus BCP group at the same time point); dotted lines indicate pharmacological treatment). (F) Enlarged graph of D from POD 38 to 44. (G) Chronic FTY720 administration attenuated BCP-induced MWT decreases on day 40. (n = 6; Kruskal-Wallis test. #: versus BCP group at the same time point); dotted lines indicate pharmacological treatment). (H) FTY720 reversed the decline in the preference of sucrose consumption induced by BCP (n = 12 one-way ANOVA, *: versus Sham group at the same time point; #: versus BCP group at the same time point). The data are presented mean ± SEM. * P <0.05, ** P <0.01, *** P <0.001; BCP, bone cancer pain; TWL, thermal withdrawal latency; MWT, mechanical withdrawal threshold; OE, overexpression; KD, knock down.

    Journal: International Journal of Biological Sciences

    Article Title: PRG-1 relieves pain and depressive-like behaviors in rats of bone cancer pain by regulation of dendritic spine in hippocampus

    doi: 10.7150/ijbs.59032

    Figure Lengend Snippet: PRG-1 attenuated pain and depression-like behaviors in BCP rats. (A) Schematic diagram of hippocampal microinjection (x = ±2.0 mm, y = -3.72 mm, and z = -3.0 mm). (B) Hippocampus injection of virus vector LV-Plppr4 (PRG-1 OE) and LV-P2X7R-RNAi (P2X 7 receptor KD) alleviated BCP-induced TWL decreases while LV-Plppr4-RNAi (PRG-1 KD) lead to early and intensified pain (n = 12; one-way ANOVA, *: versus Sham group at the same time point; #: versus BCP group at the same time point). (C) Hippocampus injection of virus vector LV-Plppr4 (PRG-1 OE) and LV-P2X7R-RNAi (P2X 7 receptor KD) alleviated BCP-induced MWT decreases while LV-Plppr4-RNAi (PRG-1 KD) lead to early and intensified pain (n = 12; one-way ANOVA, *: versus Sham group at the same time point; #: versus BCP group at the same time point). (D) PRG-1 OE reversed the preference of sucrose (n = 12; one-way ANOVA, #: versus BCP group at the same time point). (E) Multiple FTY720 administration attenuated BCP-induced thermal withdrawal latency decreases from day 39 to 43 (n = 12; one-way ANOVA, *: versus Sham group at the same time point; #: versus BCP group at the same time point); dotted lines indicate pharmacological treatment). (F) Enlarged graph of D from POD 38 to 44. (G) Chronic FTY720 administration attenuated BCP-induced MWT decreases on day 40. (n = 6; Kruskal-Wallis test. #: versus BCP group at the same time point); dotted lines indicate pharmacological treatment). (H) FTY720 reversed the decline in the preference of sucrose consumption induced by BCP (n = 12 one-way ANOVA, *: versus Sham group at the same time point; #: versus BCP group at the same time point). The data are presented mean ± SEM. * P <0.05, ** P <0.01, *** P <0.001; BCP, bone cancer pain; TWL, thermal withdrawal latency; MWT, mechanical withdrawal threshold; OE, overexpression; KD, knock down.

    Article Snippet: For immunofluorescence, the sections were blocked and permeabilized, and then incubated with goat anti-rat P2X 7 receptors antibody (1:400, abcam, USA) or rabbit anti-rat PRG-1 antibody (1:500, synaptic system).

    Techniques: Injection, Plasmid Preparation, Over Expression

    PRG-1 attenuated neuron deactivation and synaptic depression in the hippocampus induced by BCP. (A) Nissl staining in CA1, DG districts of rats' hippocampus under FTY720 or A438079 treatment. (B) The graph shows the percent of nissl positive cell. n = 6; one-way ANOVA, *: versus Sham group. (C) Images of Golgi-Cox staining and (D-F) quantitative analysis showed that the spine density depression induced by BCP was reversed by FTY720. (D, n =20 Sham, 12 BCP, 14 BCP+FTY720 and 13 BCP+A438079, one-way ANOVA; E, n = 20 Sham,11 BCP, 12 BCP+FTY720 and 12 BCP+A438079, one-way ANOVA; F, n = 12 Sham, 10 BCP, 15 BCP+FTY720 and 15 BCP+A438079, Kruskal-Wallis test; n represents analyzed dendritic segments; *: versus Sham group; #: versus BCP group); stratum radiatum (sr, apical dendrites) and stratum oriens (so, basal dendrites) of the CA1 region, and the stratum moleculare of the dentate gyrus (moDG). (G) PRG-1 OE and P2X 7 R KD rescued nissl bodies and nucleoli in CA1 and DG regions. The data are expressed as the mean ± SEM; * P <0.05, ** P <0.01, *** P <0.001, BCP, bone cancer pain; OE, overexpression; KD, knock down.

    Journal: International Journal of Biological Sciences

    Article Title: PRG-1 relieves pain and depressive-like behaviors in rats of bone cancer pain by regulation of dendritic spine in hippocampus

    doi: 10.7150/ijbs.59032

    Figure Lengend Snippet: PRG-1 attenuated neuron deactivation and synaptic depression in the hippocampus induced by BCP. (A) Nissl staining in CA1, DG districts of rats' hippocampus under FTY720 or A438079 treatment. (B) The graph shows the percent of nissl positive cell. n = 6; one-way ANOVA, *: versus Sham group. (C) Images of Golgi-Cox staining and (D-F) quantitative analysis showed that the spine density depression induced by BCP was reversed by FTY720. (D, n =20 Sham, 12 BCP, 14 BCP+FTY720 and 13 BCP+A438079, one-way ANOVA; E, n = 20 Sham,11 BCP, 12 BCP+FTY720 and 12 BCP+A438079, one-way ANOVA; F, n = 12 Sham, 10 BCP, 15 BCP+FTY720 and 15 BCP+A438079, Kruskal-Wallis test; n represents analyzed dendritic segments; *: versus Sham group; #: versus BCP group); stratum radiatum (sr, apical dendrites) and stratum oriens (so, basal dendrites) of the CA1 region, and the stratum moleculare of the dentate gyrus (moDG). (G) PRG-1 OE and P2X 7 R KD rescued nissl bodies and nucleoli in CA1 and DG regions. The data are expressed as the mean ± SEM; * P <0.05, ** P <0.01, *** P <0.001, BCP, bone cancer pain; OE, overexpression; KD, knock down.

    Article Snippet: For immunofluorescence, the sections were blocked and permeabilized, and then incubated with goat anti-rat P2X 7 receptors antibody (1:400, abcam, USA) or rabbit anti-rat PRG-1 antibody (1:500, synaptic system).

    Techniques: Staining, Over Expression

    PRG-1/PP2A interaction were increased by hippocampal injections of FTY720. (A) Images of PRG-1 (red) and P2X 7 receptor (green) in the hippocampus from BCP, BCP+FTY720 and BCP+A438079 rats by immunofluorescence on the POD 44 (scale bar = 50 μm). (B) Western blot showed that the expression of PRG-1 and P2X 7 receptor was increased in the hippocampus of BCP+FTY720 rats. (C) Co-immunoprecipitation (IP) using a PRG-1 antibody shows PRG-1 and PP2A association. (D-E) Quantitative analysis of RT-qPCR showed that the transcription of PRG-1 and P2X7 receptor mRNA was increased in the hippocampus of BCP+FTY720 rats. β-actin was included as a control. The data are expressed as the mean ± SEM; n = 6; D: Kruskal-Wallis test, E: one-way ANOVA; * P <0.05, ** P <0.005, *** P <0.001 compared to the BCP group. (F) Band of western blot showed the expression of PRG-1 and P2X 7 receptor induced by virus expression vectors. BCP, bone cancer pain; OE, overexpression; KD, knock down; POD, postoperative day.

    Journal: International Journal of Biological Sciences

    Article Title: PRG-1 relieves pain and depressive-like behaviors in rats of bone cancer pain by regulation of dendritic spine in hippocampus

    doi: 10.7150/ijbs.59032

    Figure Lengend Snippet: PRG-1/PP2A interaction were increased by hippocampal injections of FTY720. (A) Images of PRG-1 (red) and P2X 7 receptor (green) in the hippocampus from BCP, BCP+FTY720 and BCP+A438079 rats by immunofluorescence on the POD 44 (scale bar = 50 μm). (B) Western blot showed that the expression of PRG-1 and P2X 7 receptor was increased in the hippocampus of BCP+FTY720 rats. (C) Co-immunoprecipitation (IP) using a PRG-1 antibody shows PRG-1 and PP2A association. (D-E) Quantitative analysis of RT-qPCR showed that the transcription of PRG-1 and P2X7 receptor mRNA was increased in the hippocampus of BCP+FTY720 rats. β-actin was included as a control. The data are expressed as the mean ± SEM; n = 6; D: Kruskal-Wallis test, E: one-way ANOVA; * P <0.05, ** P <0.005, *** P <0.001 compared to the BCP group. (F) Band of western blot showed the expression of PRG-1 and P2X 7 receptor induced by virus expression vectors. BCP, bone cancer pain; OE, overexpression; KD, knock down; POD, postoperative day.

    Article Snippet: For immunofluorescence, the sections were blocked and permeabilized, and then incubated with goat anti-rat P2X 7 receptors antibody (1:400, abcam, USA) or rabbit anti-rat PRG-1 antibody (1:500, synaptic system).

    Techniques: Immunofluorescence, Western Blot, Expressing, Immunoprecipitation, Quantitative RT-PCR, Over Expression

    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

    Immunohistochemical staining of the dental pulp of rat. Normal group: A1 crown pulp; A2 upper 1/3 of the root; A3 middle 1/3 of the root; and A4 root tip 1/3; NS group: B1 crown pulp; B2 upper 1/3 of the root; B3 middle 1/3 of the root; and B4 root tip 1/3; LPS group: C1 crown pulp; C2 upper 1/3 of the root; C3 middle 1/3 of the root; and C4 root tip 1/3; Transverse images of the mesial root were selected for the pulp parts, the arrows indicate the P2X7 receptor expressed in the odontoblast layer in a yellowish-brown granular form. Scale bar = 50 μm.

    Journal: Molecular Pain

    Article Title: Activation of the P2X7 receptor in the dental pulp tissue contributes to the pain in rats with acute pulpitis

    doi: 10.1177/17448069221106844

    Figure Lengend Snippet: Immunohistochemical staining of the dental pulp of rat. Normal group: A1 crown pulp; A2 upper 1/3 of the root; A3 middle 1/3 of the root; and A4 root tip 1/3; NS group: B1 crown pulp; B2 upper 1/3 of the root; B3 middle 1/3 of the root; and B4 root tip 1/3; LPS group: C1 crown pulp; C2 upper 1/3 of the root; C3 middle 1/3 of the root; and C4 root tip 1/3; Transverse images of the mesial root were selected for the pulp parts, the arrows indicate the P2X7 receptor expressed in the odontoblast layer in a yellowish-brown granular form. Scale bar = 50 μm.

    Article Snippet: Escherichia coli lipopolysaccharide (LPS) was purchased from Sigma, USA (L2880, 1 mg/mL, diluted in NS); Rabbit polyclonal antibody against rat P2X7 was bought from Abcam (USA); Rabbit Hypersensitivity two-step Detection Kit, bovine serum protein, and 3,3’-diaminobenzidine (DAB) color Development Kit were acquired from Beijing Zhongshan Jinqiao; P2X7 receptor antagonist A-740003 was procured from TargetMol (USA).

    Techniques: Immunohistochemical staining, Staining

    Western Blotting bands of the P2X7 receptor protein in the dental pulp tissue of rats in each group.

    Journal: Molecular Pain

    Article Title: Activation of the P2X7 receptor in the dental pulp tissue contributes to the pain in rats with acute pulpitis

    doi: 10.1177/17448069221106844

    Figure Lengend Snippet: Western Blotting bands of the P2X7 receptor protein in the dental pulp tissue of rats in each group.

    Article Snippet: Escherichia coli lipopolysaccharide (LPS) was purchased from Sigma, USA (L2880, 1 mg/mL, diluted in NS); Rabbit polyclonal antibody against rat P2X7 was bought from Abcam (USA); Rabbit Hypersensitivity two-step Detection Kit, bovine serum protein, and 3,3’-diaminobenzidine (DAB) color Development Kit were acquired from Beijing Zhongshan Jinqiao; P2X7 receptor antagonist A-740003 was procured from TargetMol (USA).

    Techniques: Western Blot

    Comparison of the P2X7 receptor protein expression in the dental pulp tissue of rats in each group. *** p < 0.001, compared to the normal group; and ▲▲▲ p < 0.001, compared to the NS group.

    Journal: Molecular Pain

    Article Title: Activation of the P2X7 receptor in the dental pulp tissue contributes to the pain in rats with acute pulpitis

    doi: 10.1177/17448069221106844

    Figure Lengend Snippet: Comparison of the P2X7 receptor protein expression in the dental pulp tissue of rats in each group. *** p < 0.001, compared to the normal group; and ▲▲▲ p < 0.001, compared to the NS group.

    Article Snippet: Escherichia coli lipopolysaccharide (LPS) was purchased from Sigma, USA (L2880, 1 mg/mL, diluted in NS); Rabbit polyclonal antibody against rat P2X7 was bought from Abcam (USA); Rabbit Hypersensitivity two-step Detection Kit, bovine serum protein, and 3,3’-diaminobenzidine (DAB) color Development Kit were acquired from Beijing Zhongshan Jinqiao; P2X7 receptor antagonist A-740003 was procured from TargetMol (USA).

    Techniques: Expressing

    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