p2x7 receptor antagonist a 740003  (TargetMol)

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

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

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

Journal: International Journal of Molecular Sciences

Article Title: Interaction between Calcium Chelators and the Activity of P2X7 Receptors in Mouse Motor Synapses

doi: 10.3390/ijms21062034

Figure Lengend Snippet: P2X7 receptor-mediated recovery of evoked neuromuscular transmission at the NMJs of WT mice in the presence of a fast Ca 2+ chelator during a short high-frequency EPP train (50 Hz, 1 s). ( A ) Changes in the EPP quantal content in the control group (n = 24) and during the activation of P2X7 receptors by their agonist BzATP (30 μM) (n = 22). ( B ) MEPP amplitude in the control group and in the presence of BzATP. ( C ) Changes in the EPP quantal content in the control group (n = 16), after incubation with BAPTA-AM (n = 15) and during the subsequent application of BzATP (n = 19). ( D ) MEPP amplitude in the control group, after incubation with BAPTA-AM and during the subsequent application of BzATP. ( E ) The ratio of the average quantal content of the last ten EPPs in the train to the quantal content of the first EPP (EPP plateau /EPP 1 ) normalized to EPP 1 (taken as 100%) in the control group, after incubation with BAPTA-AM and during the application of BzATP in the presence of preloaded BAPTA. ( F ) Changes in the EPP quantal content in the control group (n = 22), after incubation with BAPTA-AM (n = 15) and during the subsequent application of BzATP in the presence of P2X7 receptor antagonist A740003 (1 μM) (n = 15). ( G ) MEPP amplitude in the control group, after incubation with BAPTA-AM and during the subsequent application of BzATP in the presence of A740003. ( H ) The ratio of the average quantal content of the last ten EPPs in the train to the quantal content of the first EPP (EPP plateau /EPP 1 ) normalized to EPP 1 (taken as 100%) in the control group, after incubation with BAPTA-AM and during subsequent application of BzATP in the presence of A740003. The symbols, histograms and error bars represent the mean ± SEM. * p < 0.05.

Article Snippet: The fast Ca 2+ chelator (BAPTA-AM), the slow Ca 2+ chelator (EGTA-AM) and P2X7 receptor antagonist A740003 were purchased from Tocris/Bio-Techne (Minneapolis, MN, USA) and Calbiochem Biochemicals (Nottingham, Great Britain), respectively.

Techniques: Transmission Assay, Activation Assay, Incubation

Journal: International Journal of Molecular Sciences

Article Title: Interaction between Calcium Chelators and the Activity of P2X7 Receptors in Mouse Motor Synapses

doi: 10.3390/ijms21062034

Figure Lengend Snippet: Intraterminal Ca 2+ buffering with slow Ca2+ chelator prevented P2X7 receptor-mediated potentiation of evoked neuromuscular transmission at the NMJs of pannexin-1 knockout (Panx1 −/− ) mice during a short high-frequency EPP train (50 Hz, 1 s). ( A ) Changes in the EPP quantal content at the NMJs of Panx1 −/− mice in the control group (n = 19) and during the activation of P2X7 receptors by their agonist BzATP (30 μM) (n = 15). ( B ) MEPP amplitude in the control group and upon application of BzATP. ( C ) The ratio of the average quantal content of the last ten EPPs in the train to the quantal content of the first EPP (EPP plateau /EPP 1 ) normalized to EPP 1 (taken as 100%) in the control group and during the application of BzATP. ( D ) Changes in the EPP quantal content at the NMJs of Panx1 −/− mice in the control group (n = 24), after incubation with EGTA-AM (50 µM) (n = 20) and during the application of BzATP in the presence of preloaded EGTA (n = 15). ( E ) MEPP amplitude in the control group, after incubation with EGTA-AM and during the subsequent application of BzATP. The symbols, histograms and error bars represent the mean ± SEM. * p < 0.05.

Article Snippet: The fast Ca 2+ chelator (BAPTA-AM), the slow Ca 2+ chelator (EGTA-AM) and P2X7 receptor antagonist A740003 were purchased from Tocris/Bio-Techne (Minneapolis, MN, USA) and Calbiochem Biochemicals (Nottingham, Great Britain), respectively.

Techniques: Transmission Assay, Knock-Out, Activation Assay, Incubation

Journal: International Journal of Molecular Sciences

Article Title: Interaction between Calcium Chelators and the Activity of P2X7 Receptors in Mouse Motor Synapses

doi: 10.3390/ijms21062034

Figure Lengend Snippet: The activation of P2X7 receptors restored the decrease in evoked neuromuscular transmission at the NMJs of Panx1 −/− mice induced by incubation with a fast Ca 2+ chelator. ( A ) Changes in the EPP quantal content in the control group (n = 22), after incubation with BAPTA-AM (50 µM) (n = 17) and during the subsequent activation of P2X7 receptors by their agonist BzATP (30 µM) (n = 15). ( B ) MEPP amplitude in the control group, after incubation with BAPTA-AM and during the subsequent application of BzATP. ( C ) The ratio of the average quantal content of the last ten EPPs in the train to the quantal content of the first EPP (EPP plateau /EPP 1 ) normalized to EPP 1 (taken as 100%) in the control group, after incubation with BAPTA-AM and during the application of BzATP in the presence of preloaded BAPTA. The symbols, histograms and error bars represent the mean ± SEM. * p < 0.05.

Article Snippet: The fast Ca 2+ chelator (BAPTA-AM), the slow Ca 2+ chelator (EGTA-AM) and P2X7 receptor antagonist A740003 were purchased from Tocris/Bio-Techne (Minneapolis, MN, USA) and Calbiochem Biochemicals (Nottingham, Great Britain), respectively.

Techniques: Activation Assay, Transmission Assay, Incubation

Journal: International Journal of Molecular Sciences

Article Title: Interaction between Calcium Chelators and the Activity of P2X7 Receptors in Mouse Motor Synapses

doi: 10.3390/ijms21062034

Figure Lengend Snippet: Blocking L-type voltage-dependent Ca 2+ channels (VDCCs) with nitrendipine (1 µM) failed to prevent the P2X7-mediated recovery of BAPTA-induced depression of evoked neuromuscular transmission at the NMJs of Panx1 −/− mice. ( A ) Changes in the EPP quantal content in the control group (n = 24), after incubation with BAPTA-AM (50 µM) (n = 22) and during the subsequent activation of P2X7 receptors by their agonist BzATP (30 µM) in the presence of nitrendipine (n = 21). ( B ) MEPP amplitude in the control group, after incubation with BAPTA-AM and during the subsequent application of BzATP in the presence of nitrendipine. ( C )The ratio of the average quantal content of the last ten EPPs in the train to the quantal content of the first EPP (EPP plateau /EPP 1 ) normalized to EPP 1 (taken as 100%) in the control group, after incubation with BAPTA-AM and during the application of BzATP with nitrendipine in the presence of preloaded BAPTA. The symbols, histograms and error bars represent the mean ± SEM. * p < 0.05.

Article Snippet: The fast Ca 2+ chelator (BAPTA-AM), the slow Ca 2+ chelator (EGTA-AM) and P2X7 receptor antagonist A740003 were purchased from Tocris/Bio-Techne (Minneapolis, MN, USA) and Calbiochem Biochemicals (Nottingham, Great Britain), respectively.

Techniques: Blocking Assay, Transmission Assay, Incubation, Activation Assay

Journal: International Journal of Molecular Sciences

Article Title: Interaction between Calcium Chelators and the Activity of P2X7 Receptors in Mouse Motor Synapses

doi: 10.3390/ijms21062034

Figure Lengend Snippet: Suggested contributions of various presynaptic Ca 2+ inputs, interacting with slow or fast Ca 2+ chelators, to evoked synaptic transmission in mouse NMJs. ( A ) Without Ca 2+ chelators, activation of P2X7 receptors facilitates ACh release only in NMJs of Panx1 −/− mice due to activation of CAMKII and L-type VDCCs . ( B ) In NMJs of WT mice, where L-type VDCCs are inactive, EGTA failed to change ACh release due to an inability of the slow Ca 2+ chelator to affect Ca 2+ entry via P/Q-type VDCCs. Activation of P2X7 receptors in the presence of EGTA would not induce any more changes since the stimulation of P2X7 receptors fails to disinhibit L-type VDCCs in NMJs of WT mice . In NMJs of Panx1 −/− mice, P2X7 receptor-mediated Ca 2+ entry partially decreases EGTA buffering capacity, which is still sufficient to disrupt coupling of Ca 2+ entry via L-type VDCCs to ACh release. ( C ) Both in NMJs of WT and Panx1 −/− mice, Ca 2+ entry via P2X7 receptors partially decreases BAPTA buffering capacity, leading to restoration of P/Q-type VDCC-triggered ACh release. In NMJs of Panx1 −/− mice, buffering capacity of BAPTA, even diminished by Ca2+ entry via P2X7 receptors, is still sufficient to downregulate Ca 2+ entry via L-type VDCCs, thus preventing the facilitation of ACh release. Triangles with dashed borders represent the proposed changes in the presynaptic Ca 2+ domains, which correspond to the activity of distinct Ca 2+ inputs during synaptic activity in mouse NMJs.

Article Snippet: The fast Ca 2+ chelator (BAPTA-AM), the slow Ca 2+ chelator (EGTA-AM) and P2X7 receptor antagonist A740003 were purchased from Tocris/Bio-Techne (Minneapolis, MN, USA) and Calbiochem Biochemicals (Nottingham, Great Britain), respectively.

Techniques: Transmission Assay, Activation Assay, Activity Assay

Journal: The journal of headache and pain

Article Title: Src family kinases activity is required for transmitting purinergic P2X7 receptor signaling in cortical spreading depression and neuroinflammation.

doi: 10.1186/s10194-021-01359-8

Figure Lengend Snippet: Fig. 2 Both deactivation of P2X7 receptor and SFKs reduced cortical susceptibility to CSD in mouse brain slices. a The captured images of a mouse brain slice before and after CSD induction by 260 mM KCl in cerebral cortex. The arrow indicated the direction of CSD propagation. An AOI was selected and kept the same for data analysis. b The biphasic CSD curve generated from the images recorded for 15 minutes by plotting averaged grey level within the AOI against time. CSD latency (sec) is the time interval between KCl application and CSD elicitation at the AOI. CSD propagation rate (mm/min) is the velocity by which CSD propagates along cerebral cortex. c Representative traces of the 1st peak of CSD affected by 0.03% DMSO, 3 μM A740003 and 0.5 μM saracatinib. Only the trace recorded during the first 100 seconds was displayed here in order to clearly show the starting points of KCl application and CSD elicitation. d e Effects of 0.03% DMSO, 3 μM A740003 and 0.5 μM saracatinib on CSD latency and propagation rate. Abbreviations: saracatinib (SRCT); seconds (sec); mm/minute (mm/min). Two-tailed unpaired t-test was used for comparison in CSD latency and propagation rate between DMSO and A740003 group, DMSO and saracatinib group. Significant differences were indicated by * p < 0.05, ** p < 0.01

Article Snippet: Three series of studies were designed using this ex vivo CSD model. Series 1: In order to study whether modulation of SFKs or P2X7 receptor activity could similarly regulate cortical susceptibility to CSD, the effects of a SFKs inhibitor, saracatinib (S1006, Selleckchem, Houston, USA), that is currently investigated in clinical trials for treating several neurological disorders [25, 28] or a P2X7 receptor antagonist, A740003 (3701, Tocris, Bristol, UK) on CSD latency and propagation rate were examined respectively in C57BL/6J mice brain slices.

Techniques: Slice Preparation, Generated, Two Tailed Test, Comparison

Journal: The journal of headache and pain

Article Title: Src family kinases activity is required for transmitting purinergic P2X7 receptor signaling in cortical spreading depression and neuroinflammation.

doi: 10.1186/s10194-021-01359-8

Figure Lengend Snippet: Fig. 3 Disrupting P2X7 receptor-SFKs interaction reduced cortical susceptibility to CSD and CSD-associated neuroinflammation in mouse brain slices. a Representative traces of 1st peak of CSD affected by Kreb’s, 3 μM TAT-P2X7 and TAT-P2X7SC. Only the trace recorded during the first 100 seconds was displayed here in order to clearly show CSD latency in respective group. b c Effects of Kreb’s, 3 μM TAT-P2X7 and TAT-P2X7SC on CSD latency and propagation rate. d e Effects of 3 μM TAT-P2X7 and TAT-P2X7SC on IL-1β and TNFα mRNA fold change after CSD in mouse brain slices. Two-tailed unpaired t-test was used for comparison in CSD latency and propagation rate between Kreb’s and TAT-P2X7 group, TAT- P2X7 and TAT-P2X7SC group; in IL-1β and TNFα mRNA fold change between TAT-P2X7 and TAT-P2X7SC group. Significant differences were indicated by * p < 0.05, ** p < 0.01

Article Snippet: Three series of studies were designed using this ex vivo CSD model. Series 1: In order to study whether modulation of SFKs or P2X7 receptor activity could similarly regulate cortical susceptibility to CSD, the effects of a SFKs inhibitor, saracatinib (S1006, Selleckchem, Houston, USA), that is currently investigated in clinical trials for treating several neurological disorders [25, 28] or a P2X7 receptor antagonist, A740003 (3701, Tocris, Bristol, UK) on CSD latency and propagation rate were examined respectively in C57BL/6J mice brain slices.

Techniques: Two Tailed Test, Comparison

Journal: The journal of headache and pain

Article Title: Src family kinases activity is required for transmitting purinergic P2X7 receptor signaling in cortical spreading depression and neuroinflammation.

doi: 10.1186/s10194-021-01359-8

Figure Lengend Snippet: Fig. 4 NMDA restored the disrupted P2X7 receptor-SFKs interaction-reduced cortical susceptibility to CSD but not CSD-associated neuroinflammation in mouse brain slices. a Effects of 3 μM TAT-P2X7 and TAT-P2X7SC on glutamate release from mouse brain slices after CSD. b Representative traces of 1st peak of CSD affected by 3 μM TAT-P2X7 and 3 μM TAT-P2X7 + 10 μM NMDA. c d Effects of 3 μM TAT-P2X7 + 10 μM NMDA on CSD latency and propagation rate. e Effects of 3 μM TAT-P2X7 + 10 μM NMDA on CSD-associated IL-1β mRNA fold change in mouse brain slices. Two-tailed unpaired t- test was used for comparison in glutamate release between TAT-P2X7 and TAT-P2X7SC group; in CSD latency, propagation rate and IL-1β mRNA fold change between TAT-P2X7 and TAT-P2X7 + NMDA group. Significant differences were indicated by * p < 0.05

Article Snippet: Three series of studies were designed using this ex vivo CSD model. Series 1: In order to study whether modulation of SFKs or P2X7 receptor activity could similarly regulate cortical susceptibility to CSD, the effects of a SFKs inhibitor, saracatinib (S1006, Selleckchem, Houston, USA), that is currently investigated in clinical trials for treating several neurological disorders [25, 28] or a P2X7 receptor antagonist, A740003 (3701, Tocris, Bristol, UK) on CSD latency and propagation rate were examined respectively in C57BL/6J mice brain slices.

Techniques: Two Tailed Test, Comparison

Journal: The journal of headache and pain

Article Title: Src family kinases activity is required for transmitting purinergic P2X7 receptor signaling in cortical spreading depression and neuroinflammation.

doi: 10.1186/s10194-021-01359-8

Figure Lengend Snippet: Fig. 6 Schematic representation of the role that P2X7 receptor/SFKs signaling may exert in CSD-associated migraine pathophysiology. P2X7 receptor/SFKs signaling is activated during CSD to facilitate neuroinflammation, NMDA receptor activation and glutamate release. Glutamate may in return reinforce the activation of NMDA receptor (dotted line with arrow), both of which facilitate cortical susceptibility to CSD, forming a positive loop

Article Snippet: Three series of studies were designed using this ex vivo CSD model. Series 1: In order to study whether modulation of SFKs or P2X7 receptor activity could similarly regulate cortical susceptibility to CSD, the effects of a SFKs inhibitor, saracatinib (S1006, Selleckchem, Houston, USA), that is currently investigated in clinical trials for treating several neurological disorders [25, 28] or a P2X7 receptor antagonist, A740003 (3701, Tocris, Bristol, UK) on CSD latency and propagation rate were examined respectively in C57BL/6J mice brain slices.

Techniques: Activation Assay

Journal: Frontiers in immunology

Article Title: Human P2Y 11 Expression Level Affects Human P2X7 Receptor-Mediated Cell Death.

doi: 10.3389/fimmu.2018.01159

Figure Lengend Snippet: Figure 1 | CD4+ and CD8+ T lymphocytes show a differential response to P2X7 stimulation. (A) The difference in CD4+ (orange rhomb) and CD8+ (purple square) T lymphocyte P2X7 receptor activation with BzATP was measured as lactate dehydrogenase (LDH) activity in the supernatant following 2 h stimulation. Data are shown as mean ± SEM normalized to maximum induced cell death in 1% Triton X-100, n = 5–6 from three independent donors. (B) The BzATP response in CD4+ and CD8+ T lymphocytes blocked by P2X7 receptor antagonist, A740003. Cells were stimulated with 1,000 µM BzATP in combination with various concentrations of P2X7 receptor antagonist, A740003, and P2X7 receptor activation was measured as the activity of LDH after 2 h. Data are shown as mean ± SEM of maximum induced cell death at 1% Triton X-100, n = 11–14 from seven independent donors. LDH baseline activity is shown as solid lines for CD4+ (orange) and CD8+ (purple) T lymphocytes, respectively.

Article Snippet: Cells were washed in PBS and loosened with versene and trypsin and cultured overnight as 3 × 105 cells/well in a black clear-bottomed poly-d-lysine-coated 96-well plate before further analysis. cell Death by lDh Measurement Cells were incubated with the synthetic ATP-analog 3'-O-(4benzoyl)benzoyl-ATP (BzATP, #B6396-25MG, Sigma) dissolved in water, P2X7 receptor antagonist A740003 (#sc-291774, Santa Cruz Biotechnology) dissolved in ethanol, 4,4'-[carbonylbis(imino3,1-(4-methyl-phenylene)-carbonylimino)]-bis(naphthalene2,6-disulfonic acid) tetrasodium salt (NF340) dissolved in water (#3830/10, Tocris) or 1% Triton X-100 (#T9284-500 mL, SigmaAldrich) for 2 h before LDH measurement.

Techniques: Activation Assay, Activity Assay

Journal: Frontiers in immunology

Article Title: Human P2Y 11 Expression Level Affects Human P2X7 Receptor-Mediated Cell Death.

doi: 10.3389/fimmu.2018.01159

Figure Lengend Snippet: Figure 2 | P2X7 levels are equally high in CD4+ and CD8+ T lymphocytes. (A) P2RX7 gene expression in human CD4+ and CD8+ T lymphocytes shown as ΔCt values normalized to the genes for GAPDH and ACTB (β-actin) from 18 to 21 independent donors. (B) Western blot showing P2X7 receptor signal (75 kDa) from protein lysate of CD4+ (orange) and CD8+ (purple) T lymphocytes from five different donors (#1–5). Loading control was β-actin (42 kDa).

Article Snippet: Cells were washed in PBS and loosened with versene and trypsin and cultured overnight as 3 × 105 cells/well in a black clear-bottomed poly-d-lysine-coated 96-well plate before further analysis. cell Death by lDh Measurement Cells were incubated with the synthetic ATP-analog 3'-O-(4benzoyl)benzoyl-ATP (BzATP, #B6396-25MG, Sigma) dissolved in water, P2X7 receptor antagonist A740003 (#sc-291774, Santa Cruz Biotechnology) dissolved in ethanol, 4,4'-[carbonylbis(imino3,1-(4-methyl-phenylene)-carbonylimino)]-bis(naphthalene2,6-disulfonic acid) tetrasodium salt (NF340) dissolved in water (#3830/10, Tocris) or 1% Triton X-100 (#T9284-500 mL, SigmaAldrich) for 2 h before LDH measurement.

Techniques: Gene Expression, Western Blot, Control

Journal: Frontiers in immunology

Article Title: Human P2Y 11 Expression Level Affects Human P2X7 Receptor-Mediated Cell Death.

doi: 10.3389/fimmu.2018.01159

Figure Lengend Snippet: Figure 3 | Higher P2Y11 levels in CD8+ T lymphocytes and immune-activated CD4+ T lymphocytes correlate with lower responses to BzATP compared to naïve CD4+ T lymphocytes. (A) Left axis: P2RY11 gene expression in human CD4+ and CD8+ T lymphocytes shown as ΔCt values normalized to the genes for GAPDH and ACTB (β-actin) from 14 to 18 independent donors. Right axis: mean fluorescence intensity (MFI) as measured by flow cytometry on eight independent donors. (B) Western blot membrane with protein lysate of CD4+ (orange) and CD8+ (purple) T lymphocytes from five different donors (#1–5) following stripping and re-blotting for P2Y11 signal (33 kDa). Loading control was β-actin (42 kDa) after stripping and re-blotting. (C) ΔCt values for human P2RY11 (green) and P2RX7 (red) gene expression over time in naïve and immune-activated CD4+ T lymphocytes. Values were normalized to the genes for B2M and EEF1A1 from three independent donors. (D) Lactate dehydrogenase (LDH) activity in the supernatant following 2 h incubation with various concentrations of BzATP of 2-day cultured naïve (filled rhomb) or immune-activated (open rhomb) CD4+ T lymphocytes. Data are shown as mean ± SEM of maximum induced cell death at 1% Triton X-100, n = 4–6 from three independent donors.

Article Snippet: Cells were washed in PBS and loosened with versene and trypsin and cultured overnight as 3 × 105 cells/well in a black clear-bottomed poly-d-lysine-coated 96-well plate before further analysis. cell Death by lDh Measurement Cells were incubated with the synthetic ATP-analog 3'-O-(4benzoyl)benzoyl-ATP (BzATP, #B6396-25MG, Sigma) dissolved in water, P2X7 receptor antagonist A740003 (#sc-291774, Santa Cruz Biotechnology) dissolved in ethanol, 4,4'-[carbonylbis(imino3,1-(4-methyl-phenylene)-carbonylimino)]-bis(naphthalene2,6-disulfonic acid) tetrasodium salt (NF340) dissolved in water (#3830/10, Tocris) or 1% Triton X-100 (#T9284-500 mL, SigmaAldrich) for 2 h before LDH measurement.

Techniques: Gene Expression, Fluorescence, Flow Cytometry, Western Blot, Membrane, Stripping Membranes, Control, Activity Assay, Incubation, Cell Culture

Journal: Frontiers in immunology

Article Title: Human P2Y 11 Expression Level Affects Human P2X7 Receptor-Mediated Cell Death.

doi: 10.3389/fimmu.2018.01159

Figure Lengend Snippet: Figure 5 | P2X7-mediated cell death and pore formation are lower when cells are cotransfected with P2RY11. Top row: the activity of lactate dehydrogenase (LDH) in the supernatant after 2 h in human embryonic kidney-293 (HEK-293) cells transiently transfected with vectors expressing purinergic receptors. Cells were stimulated with (A) increasing doses of BzATP, n = 5–6 from three experimental runs or (B) 1,000 µM BzATP in combination with various concentrations of P2X7 receptor antagonist, A740003, n = 6–8 from five experimental runs. Data are shown as mean ± SEM of maximum-induced cell death by 1% Triton X-100. LDH baseline activity is shown as solid lines for eGFP + empty vector (black), P2RX7 + empty vector (red), and P2RX7 + P2RY11 (blue), respectively. Bottom row: GripTite cells transiently transfected with vectors expressing purinergic receptors were stimulated with various concentrations of BzATP to induce pore formation measured as YO-PRO-1 fluorescent dye uptake. Cells were stimulated with (C) increasing doses of BzATP, n = 12–29 from nine experimental runs or (D) BzATP and P2X7 receptor antagonist, A740003, n = 6–15 from five experimental runs. Data are shown as mean ± SEM.

Article Snippet: Cells were washed in PBS and loosened with versene and trypsin and cultured overnight as 3 × 105 cells/well in a black clear-bottomed poly-d-lysine-coated 96-well plate before further analysis. cell Death by lDh Measurement Cells were incubated with the synthetic ATP-analog 3'-O-(4benzoyl)benzoyl-ATP (BzATP, #B6396-25MG, Sigma) dissolved in water, P2X7 receptor antagonist A740003 (#sc-291774, Santa Cruz Biotechnology) dissolved in ethanol, 4,4'-[carbonylbis(imino3,1-(4-methyl-phenylene)-carbonylimino)]-bis(naphthalene2,6-disulfonic acid) tetrasodium salt (NF340) dissolved in water (#3830/10, Tocris) or 1% Triton X-100 (#T9284-500 mL, SigmaAldrich) for 2 h before LDH measurement.

Techniques: Activity Assay, Transfection, Expressing, Plasmid Preparation

Journal: Frontiers in immunology

Article Title: Human P2Y 11 Expression Level Affects Human P2X7 Receptor-Mediated Cell Death.

doi: 10.3389/fimmu.2018.01159

Figure Lengend Snippet: Figure 6 | P2X7-mediated Ca2+ response is still present when cells are cotransfected with P2RY11. Normalized calcium response measured by Fluo-4 following stimulation with various concentrations of BzATP of transiently transfected GripTite cells. Data are shown as mean ± SD, n = 9 from three independent experiments. Dose–response curves for P2RX7 (red) and P2RY11 (green) were fitted to a sigmoidal curve and cotransfected P2RX7 + P2RY11 to a biphasic sigmoidal curve. Vertical dashed lines represent EC50 values for respective curves and 95% confidence intervals in colored shades. See text for details.

Article Snippet: Cells were washed in PBS and loosened with versene and trypsin and cultured overnight as 3 × 105 cells/well in a black clear-bottomed poly-d-lysine-coated 96-well plate before further analysis. cell Death by lDh Measurement Cells were incubated with the synthetic ATP-analog 3'-O-(4benzoyl)benzoyl-ATP (BzATP, #B6396-25MG, Sigma) dissolved in water, P2X7 receptor antagonist A740003 (#sc-291774, Santa Cruz Biotechnology) dissolved in ethanol, 4,4'-[carbonylbis(imino3,1-(4-methyl-phenylene)-carbonylimino)]-bis(naphthalene2,6-disulfonic acid) tetrasodium salt (NF340) dissolved in water (#3830/10, Tocris) or 1% Triton X-100 (#T9284-500 mL, SigmaAldrich) for 2 h before LDH measurement.

Techniques: Transfection

Journal: Frontiers in immunology

Article Title: Human P2Y 11 Expression Level Affects Human P2X7 Receptor-Mediated Cell Death.

doi: 10.3389/fimmu.2018.01159

Figure Lengend Snippet: Figure 7 | The effect of P2Y11 on P2X7-mediated pore formation is not dependent on P2Y11 signaling. Stimulation with various concentrations of BzATP of transiently transfected GripTite cells expressing empty vector (black), P2RY11 (closed green square), or P2RY11 C919T (open green square) vector in (A) calcium assay measured by Fluo-4, data are shown as mean ± SEM, n = 3,6 from 1 to 2 independent runs. (B) cAMP response, data are shown as mean ± SEM, n = 3,6 from 1 to 2 independent runs. (C) YO-PRO-1 dye uptake as a measure of P2X7 receptor pore formation in cells transfected with P2RX7 + empty vector (red), P2RX7 + P2RY11 (half-open blue triangle), and P2RX7 + P2RY11 C919T (open blue triangle). Data are shown as mean ± SEM, n = 9 from three independent runs. Part of data was previously published by Ref. (25).

Article Snippet: Cells were washed in PBS and loosened with versene and trypsin and cultured overnight as 3 × 105 cells/well in a black clear-bottomed poly-d-lysine-coated 96-well plate before further analysis. cell Death by lDh Measurement Cells were incubated with the synthetic ATP-analog 3'-O-(4benzoyl)benzoyl-ATP (BzATP, #B6396-25MG, Sigma) dissolved in water, P2X7 receptor antagonist A740003 (#sc-291774, Santa Cruz Biotechnology) dissolved in ethanol, 4,4'-[carbonylbis(imino3,1-(4-methyl-phenylene)-carbonylimino)]-bis(naphthalene2,6-disulfonic acid) tetrasodium salt (NF340) dissolved in water (#3830/10, Tocris) or 1% Triton X-100 (#T9284-500 mL, SigmaAldrich) for 2 h before LDH measurement.

Techniques: Transfection, Expressing, Plasmid Preparation, Calcium Assay