anti purinergic p2x 7 r antibody  (Alomone Labs)


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    Structured Review

    Alomone Labs anti purinergic p2x 7 r antibody
    (A). Astrocytes were treated with PACAP (20 ng/ml), or indirectly co-cultured with hMSCs in the absence and presence of PACAP (20 ng/ml) for 48 hours. The cultures were subjected to Q-PCR for measurement of the mRNA levels of GLT-1 and GLAST. Data are presented as mean ± SEM and expressed as the ratio of GLT-1 (GLAST) mRNA levels compared to control. * p <0.05 versus control. (B). Astrocytes were indirectly co-cultured with hMSCs in the absence or presence of PACAP for 48 hours. Membrane proteins were then extracted for western blotting using anti-GLT-1 antibody. The same blot was reprobed with <t>anti-P2X</t> 7 R antibody. <t>P2X</t> <t>7</t> <t>R</t> levels are presented as a loading control. Relative intensity of GLT-1 levels normalized to P2X 7 R was measured. Data are presented as mean ± SEM and expressed as a percentage of GLT-1 levels in the group with combinatorial treatment compared to that detected in the group co-cultured only with hMSCs. * p <0.05 versus the group only co-cultured with hMSCs. (C). Astrocytes were infected by lentivirus carrying lenti-GLT-1-GFP, and then co-cultured with hMSCs in the absence or presence of PACAP. Strong punctate fluorescence spots (arrowheads) were observed in the processes of astrocytes co-cultured with hMSCs in the presence of PACAP, when compared to that seen in astrocytes co-cultured only with hMSCs (arrows). Scale bar, 50 µm. (D). Astrocytes were treated with PACAP, or co-cultured with hMSCs in the absence or presence of PACAP. After 48 or 72 hours, the cultures were subjected to [ 3 H]-L-glutamate uptake analysis as described in . Data are presented as mean ± SEM and expressed as a ratio of the glutamate uptake in each treated group compared to that detected in the control group. * p <0.05 versus control.
    Anti Purinergic P2x 7 R 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/anti purinergic p2x 7 r antibody/product/Alomone Labs
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti purinergic p2x 7 r antibody - by Bioz Stars, 2023-02
    94/100 stars

    Images

    1) Product Images from "Effects of Combinatorial Treatment with Pituitary Adenylate Cyclase Activating Peptide and Human Mesenchymal Stem Cells on Spinal Cord Tissue Repair"

    Article Title: Effects of Combinatorial Treatment with Pituitary Adenylate Cyclase Activating Peptide and Human Mesenchymal Stem Cells on Spinal Cord Tissue Repair

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0015299

    (A). Astrocytes were treated with PACAP (20 ng/ml), or indirectly co-cultured with hMSCs in the absence and presence of PACAP (20 ng/ml) for 48 hours. The cultures were subjected to Q-PCR for measurement of the mRNA levels of GLT-1 and GLAST. Data are presented as mean ± SEM and expressed as the ratio of GLT-1 (GLAST) mRNA levels compared to control. * p <0.05 versus control. (B). Astrocytes were indirectly co-cultured with hMSCs in the absence or presence of PACAP for 48 hours. Membrane proteins were then extracted for western blotting using anti-GLT-1 antibody. The same blot was reprobed with anti-P2X 7 R antibody. P2X 7 R levels are presented as a loading control. Relative intensity of GLT-1 levels normalized to P2X 7 R was measured. Data are presented as mean ± SEM and expressed as a percentage of GLT-1 levels in the group with combinatorial treatment compared to that detected in the group co-cultured only with hMSCs. * p <0.05 versus the group only co-cultured with hMSCs. (C). Astrocytes were infected by lentivirus carrying lenti-GLT-1-GFP, and then co-cultured with hMSCs in the absence or presence of PACAP. Strong punctate fluorescence spots (arrowheads) were observed in the processes of astrocytes co-cultured with hMSCs in the presence of PACAP, when compared to that seen in astrocytes co-cultured only with hMSCs (arrows). Scale bar, 50 µm. (D). Astrocytes were treated with PACAP, or co-cultured with hMSCs in the absence or presence of PACAP. After 48 or 72 hours, the cultures were subjected to [ 3 H]-L-glutamate uptake analysis as described in . Data are presented as mean ± SEM and expressed as a ratio of the glutamate uptake in each treated group compared to that detected in the control group. * p <0.05 versus control.
    Figure Legend Snippet: (A). Astrocytes were treated with PACAP (20 ng/ml), or indirectly co-cultured with hMSCs in the absence and presence of PACAP (20 ng/ml) for 48 hours. The cultures were subjected to Q-PCR for measurement of the mRNA levels of GLT-1 and GLAST. Data are presented as mean ± SEM and expressed as the ratio of GLT-1 (GLAST) mRNA levels compared to control. * p <0.05 versus control. (B). Astrocytes were indirectly co-cultured with hMSCs in the absence or presence of PACAP for 48 hours. Membrane proteins were then extracted for western blotting using anti-GLT-1 antibody. The same blot was reprobed with anti-P2X 7 R antibody. P2X 7 R levels are presented as a loading control. Relative intensity of GLT-1 levels normalized to P2X 7 R was measured. Data are presented as mean ± SEM and expressed as a percentage of GLT-1 levels in the group with combinatorial treatment compared to that detected in the group co-cultured only with hMSCs. * p <0.05 versus the group only co-cultured with hMSCs. (C). Astrocytes were infected by lentivirus carrying lenti-GLT-1-GFP, and then co-cultured with hMSCs in the absence or presence of PACAP. Strong punctate fluorescence spots (arrowheads) were observed in the processes of astrocytes co-cultured with hMSCs in the presence of PACAP, when compared to that seen in astrocytes co-cultured only with hMSCs (arrows). Scale bar, 50 µm. (D). Astrocytes were treated with PACAP, or co-cultured with hMSCs in the absence or presence of PACAP. After 48 or 72 hours, the cultures were subjected to [ 3 H]-L-glutamate uptake analysis as described in . Data are presented as mean ± SEM and expressed as a ratio of the glutamate uptake in each treated group compared to that detected in the control group. * p <0.05 versus control.

    Techniques Used: Cell Culture, Western Blot, Infection, Fluorescence

    anti purinergic p2x 7 r antibody  (Alomone Labs)


    Bioz Verified Symbol Alomone Labs is a verified supplier
    Bioz Manufacturer Symbol Alomone Labs manufactures this product  
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    Structured Review

    Alomone Labs anti purinergic p2x 7 r antibody
    (A). Astrocytes were treated with PACAP (20 ng/ml), or indirectly co-cultured with hMSCs in the absence and presence of PACAP (20 ng/ml) for 48 hours. The cultures were subjected to Q-PCR for measurement of the mRNA levels of GLT-1 and GLAST. Data are presented as mean ± SEM and expressed as the ratio of GLT-1 (GLAST) mRNA levels compared to control. * p <0.05 versus control. (B). Astrocytes were indirectly co-cultured with hMSCs in the absence or presence of PACAP for 48 hours. Membrane proteins were then extracted for western blotting using anti-GLT-1 antibody. The same blot was reprobed with <t>anti-P2X</t> 7 R antibody. <t>P2X</t> <t>7</t> <t>R</t> levels are presented as a loading control. Relative intensity of GLT-1 levels normalized to P2X 7 R was measured. Data are presented as mean ± SEM and expressed as a percentage of GLT-1 levels in the group with combinatorial treatment compared to that detected in the group co-cultured only with hMSCs. * p <0.05 versus the group only co-cultured with hMSCs. (C). Astrocytes were infected by lentivirus carrying lenti-GLT-1-GFP, and then co-cultured with hMSCs in the absence or presence of PACAP. Strong punctate fluorescence spots (arrowheads) were observed in the processes of astrocytes co-cultured with hMSCs in the presence of PACAP, when compared to that seen in astrocytes co-cultured only with hMSCs (arrows). Scale bar, 50 µm. (D). Astrocytes were treated with PACAP, or co-cultured with hMSCs in the absence or presence of PACAP. After 48 or 72 hours, the cultures were subjected to [ 3 H]-L-glutamate uptake analysis as described in . Data are presented as mean ± SEM and expressed as a ratio of the glutamate uptake in each treated group compared to that detected in the control group. * p <0.05 versus control.
    Anti Purinergic P2x 7 R 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/anti purinergic p2x 7 r antibody/product/Alomone Labs
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti purinergic p2x 7 r antibody - by Bioz Stars, 2023-02
    94/100 stars

    Images

    1) Product Images from "Effects of Combinatorial Treatment with Pituitary Adenylate Cyclase Activating Peptide and Human Mesenchymal Stem Cells on Spinal Cord Tissue Repair"

    Article Title: Effects of Combinatorial Treatment with Pituitary Adenylate Cyclase Activating Peptide and Human Mesenchymal Stem Cells on Spinal Cord Tissue Repair

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0015299

    (A). Astrocytes were treated with PACAP (20 ng/ml), or indirectly co-cultured with hMSCs in the absence and presence of PACAP (20 ng/ml) for 48 hours. The cultures were subjected to Q-PCR for measurement of the mRNA levels of GLT-1 and GLAST. Data are presented as mean ± SEM and expressed as the ratio of GLT-1 (GLAST) mRNA levels compared to control. * p <0.05 versus control. (B). Astrocytes were indirectly co-cultured with hMSCs in the absence or presence of PACAP for 48 hours. Membrane proteins were then extracted for western blotting using anti-GLT-1 antibody. The same blot was reprobed with anti-P2X 7 R antibody. P2X 7 R levels are presented as a loading control. Relative intensity of GLT-1 levels normalized to P2X 7 R was measured. Data are presented as mean ± SEM and expressed as a percentage of GLT-1 levels in the group with combinatorial treatment compared to that detected in the group co-cultured only with hMSCs. * p <0.05 versus the group only co-cultured with hMSCs. (C). Astrocytes were infected by lentivirus carrying lenti-GLT-1-GFP, and then co-cultured with hMSCs in the absence or presence of PACAP. Strong punctate fluorescence spots (arrowheads) were observed in the processes of astrocytes co-cultured with hMSCs in the presence of PACAP, when compared to that seen in astrocytes co-cultured only with hMSCs (arrows). Scale bar, 50 µm. (D). Astrocytes were treated with PACAP, or co-cultured with hMSCs in the absence or presence of PACAP. After 48 or 72 hours, the cultures were subjected to [ 3 H]-L-glutamate uptake analysis as described in . Data are presented as mean ± SEM and expressed as a ratio of the glutamate uptake in each treated group compared to that detected in the control group. * p <0.05 versus control.
    Figure Legend Snippet: (A). Astrocytes were treated with PACAP (20 ng/ml), or indirectly co-cultured with hMSCs in the absence and presence of PACAP (20 ng/ml) for 48 hours. The cultures were subjected to Q-PCR for measurement of the mRNA levels of GLT-1 and GLAST. Data are presented as mean ± SEM and expressed as the ratio of GLT-1 (GLAST) mRNA levels compared to control. * p <0.05 versus control. (B). Astrocytes were indirectly co-cultured with hMSCs in the absence or presence of PACAP for 48 hours. Membrane proteins were then extracted for western blotting using anti-GLT-1 antibody. The same blot was reprobed with anti-P2X 7 R antibody. P2X 7 R levels are presented as a loading control. Relative intensity of GLT-1 levels normalized to P2X 7 R was measured. Data are presented as mean ± SEM and expressed as a percentage of GLT-1 levels in the group with combinatorial treatment compared to that detected in the group co-cultured only with hMSCs. * p <0.05 versus the group only co-cultured with hMSCs. (C). Astrocytes were infected by lentivirus carrying lenti-GLT-1-GFP, and then co-cultured with hMSCs in the absence or presence of PACAP. Strong punctate fluorescence spots (arrowheads) were observed in the processes of astrocytes co-cultured with hMSCs in the presence of PACAP, when compared to that seen in astrocytes co-cultured only with hMSCs (arrows). Scale bar, 50 µm. (D). Astrocytes were treated with PACAP, or co-cultured with hMSCs in the absence or presence of PACAP. After 48 or 72 hours, the cultures were subjected to [ 3 H]-L-glutamate uptake analysis as described in . Data are presented as mean ± SEM and expressed as a ratio of the glutamate uptake in each treated group compared to that detected in the control group. * p <0.05 versus control.

    Techniques Used: Cell Culture, Western Blot, Infection, Fluorescence

    rabbit anti p2x7r monoclonal igg  (Alomone Labs)


    Bioz Verified Symbol Alomone Labs is a verified supplier
    Bioz Manufacturer Symbol Alomone Labs manufactures this product  
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  • 94

    Structured Review

    Alomone Labs rabbit anti p2x7r monoclonal igg
    The sequences of oligonucleotide primers.
    Rabbit Anti P2x7r Monoclonal Igg, 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 p2x7r monoclonal igg/product/Alomone Labs
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rabbit anti p2x7r monoclonal igg - by Bioz Stars, 2023-02
    94/100 stars

    Images

    1) Product Images from "The Actions and Mechanisms of P2X7R and p38 MAPK Activation in Mediating Bortezomib-Induced Neuropathic Pain"

    Article Title: The Actions and Mechanisms of P2X7R and p38 MAPK Activation in Mediating Bortezomib-Induced Neuropathic Pain

    Journal: BioMed Research International

    doi: 10.1155/2020/8143754

    The sequences of oligonucleotide primers.
    Figure Legend Snippet: The sequences of oligonucleotide primers.

    Techniques Used:

    The antibodies for immunoblotting.
    Figure Legend Snippet: The antibodies for immunoblotting.

    Techniques Used: Western Blot, Concentration Assay

    The antibodies for fluorescence labeling.
    Figure Legend Snippet: The antibodies for fluorescence labeling.

    Techniques Used: Fluorescence, Labeling, Concentration Assay

    Mechanical threshold and P2X7R and p-p38 expression. (a) Mechanical threshold after BTZ injection. (b, c) Western blot for P2X7R expression after BTZ treatment. (d) Immunofluorescence location of P2X7R in DRG. The arrows indicate the typical single- or double-labeled DRG neurons and satellite cells. P2X7R is not expressed in NF-200-positive neurons. P2X7R is expressed in GFAP-labeled satellite glial cells (SGCs). (e) Immunofluorescence location of p-p38 in DRG. The arrows indicate the typical single- or double-labeled DRG neurons and satellite cells. p-p38 is expressed in both MAP2-labeled neurons and GFAP-labeled SGCs. (f) Immunofluorescence location of P2X7R in SDH. The arrows indicate the typical single-labeled and double-labeled cells in SDH. P2X7R is expressed mainly in Iba-1-labeled microglial cells rather than in GFAP-labeled astrocytes and MAP2-labeled neurons. (g) Immunofluorescence location of p-p38 in SDH. The arrows indicate the typical single-labeled and double-labeled cells in SDH. p-p38 is expressed mainly in Iba-1-labeled microglial cells. Scale bar = 50 μ m. Mean ± SEM ( n = 5). ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001.
    Figure Legend Snippet: Mechanical threshold and P2X7R and p-p38 expression. (a) Mechanical threshold after BTZ injection. (b, c) Western blot for P2X7R expression after BTZ treatment. (d) Immunofluorescence location of P2X7R in DRG. The arrows indicate the typical single- or double-labeled DRG neurons and satellite cells. P2X7R is not expressed in NF-200-positive neurons. P2X7R is expressed in GFAP-labeled satellite glial cells (SGCs). (e) Immunofluorescence location of p-p38 in DRG. The arrows indicate the typical single- or double-labeled DRG neurons and satellite cells. p-p38 is expressed in both MAP2-labeled neurons and GFAP-labeled SGCs. (f) Immunofluorescence location of P2X7R in SDH. The arrows indicate the typical single-labeled and double-labeled cells in SDH. P2X7R is expressed mainly in Iba-1-labeled microglial cells rather than in GFAP-labeled astrocytes and MAP2-labeled neurons. (g) Immunofluorescence location of p-p38 in SDH. The arrows indicate the typical single-labeled and double-labeled cells in SDH. p-p38 is expressed mainly in Iba-1-labeled microglial cells. Scale bar = 50 μ m. Mean ± SEM ( n = 5). ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001.

    Techniques Used: Expressing, Injection, Western Blot, Immunofluorescence, Labeling

    p38 mRNA expression and p38 phosphorylation in DRG after inhibition of P2X7R with BBG. (a) p38 mRNA levels. (b) p-p38 protein immunoblotting bands. (c) p-p38 protein levels. (d) p-p38 immunofluorescence labeling. The arrows show the typical p-p38 single-labeled DRG cells. (e) p-p38 fluorescence density. Scale bar = 50 μ m. Mean ± SEM ( n = 5). ∗ P < 0.05; ∗∗∗ P < 0.001.
    Figure Legend Snippet: p38 mRNA expression and p38 phosphorylation in DRG after inhibition of P2X7R with BBG. (a) p38 mRNA levels. (b) p-p38 protein immunoblotting bands. (c) p-p38 protein levels. (d) p-p38 immunofluorescence labeling. The arrows show the typical p-p38 single-labeled DRG cells. (e) p-p38 fluorescence density. Scale bar = 50 μ m. Mean ± SEM ( n = 5). ∗ P < 0.05; ∗∗∗ P < 0.001.

    Techniques Used: Expressing, Inhibition, Western Blot, Immunofluorescence, Labeling, Fluorescence

    p38 mRNA expression and p38 phosphorylation in SDH after inhibition of P2X7R with BBG. (a) p38 mRNA levels. (b) p-p38 protein immunoblotting bands. (c) p-p38 protein levels. (d) P2X7R and p-p38 coexpression fluorescence labeling. The arrows indicate the typical single-labeled and double-labeled SDH microglia. (e) P2X7R and p-p38 coexpression fluorescence density. Scale bar = 50 μ m. Mean ± SEM ( n = 5). ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001.
    Figure Legend Snippet: p38 mRNA expression and p38 phosphorylation in SDH after inhibition of P2X7R with BBG. (a) p38 mRNA levels. (b) p-p38 protein immunoblotting bands. (c) p-p38 protein levels. (d) P2X7R and p-p38 coexpression fluorescence labeling. The arrows indicate the typical single-labeled and double-labeled SDH microglia. (e) P2X7R and p-p38 coexpression fluorescence density. Scale bar = 50 μ m. Mean ± SEM ( n = 5). ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001.

    Techniques Used: Expressing, Inhibition, Western Blot, Fluorescence, Labeling

    IL-1 β , IL-6, and TNF- α mRNA expression in DRG and SDH after inhibition of P2X7R. (a) DRG IL-1 β mRNA. (b) DRG IL-6 mRNA. (c) DRG TNF- α mRNA. (d) SDH IL-1 β mRNA. (e) SDH IL-6 mRNA. (f) SDH TNF- α mRNA. Mean ± SEM ( n = 5). ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001.
    Figure Legend Snippet: IL-1 β , IL-6, and TNF- α mRNA expression in DRG and SDH after inhibition of P2X7R. (a) DRG IL-1 β mRNA. (b) DRG IL-6 mRNA. (c) DRG TNF- α mRNA. (d) SDH IL-1 β mRNA. (e) SDH IL-6 mRNA. (f) SDH TNF- α mRNA. Mean ± SEM ( n = 5). ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001.

    Techniques Used: Expressing, Inhibition

    P2X7R mRNA and protein expression in DRG after inhibition of p38 phosphorylation. (a) P2X7R mRNA levels. (b) P2X7R protein immunoblotting bands. (c) P2X7R protein levels. (d) P2X7R and GFAP coexpression fluorescence labeling for SGCs. The arrows indicate the typical single-labeled and double-labeled DRG satellite cells. (e) P2X7R and GFAP coexpression fluorescence density. Scale bar = 50 μ m. Mean ± SEM ( n = 5). ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001.
    Figure Legend Snippet: P2X7R mRNA and protein expression in DRG after inhibition of p38 phosphorylation. (a) P2X7R mRNA levels. (b) P2X7R protein immunoblotting bands. (c) P2X7R protein levels. (d) P2X7R and GFAP coexpression fluorescence labeling for SGCs. The arrows indicate the typical single-labeled and double-labeled DRG satellite cells. (e) P2X7R and GFAP coexpression fluorescence density. Scale bar = 50 μ m. Mean ± SEM ( n = 5). ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001.

    Techniques Used: Expressing, Inhibition, Western Blot, Fluorescence, Labeling

    P2X7R mRNA and protein expression in SDH after inhibition of p38 phosphorylation. (a) P2X7R mRNA levels. (b) P2X7R protein immunoblotting bands. (c) P2X7R protein levels. (d) P2X7R and p-p38 coexpression fluorescence labeling. The arrows indicate the typical single-labeled and double-labeled SDH microglia. (e) P2X7R and p-p38 colocalization fluorescence density. Scale bar = 50 μ m. Mean ± SEM ( n = 5). ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001.
    Figure Legend Snippet: P2X7R mRNA and protein expression in SDH after inhibition of p38 phosphorylation. (a) P2X7R mRNA levels. (b) P2X7R protein immunoblotting bands. (c) P2X7R protein levels. (d) P2X7R and p-p38 coexpression fluorescence labeling. The arrows indicate the typical single-labeled and double-labeled SDH microglia. (e) P2X7R and p-p38 colocalization fluorescence density. Scale bar = 50 μ m. Mean ± SEM ( n = 5). ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001.

    Techniques Used: Expressing, Inhibition, Western Blot, Fluorescence, Labeling

    Mechanical threshold alterations after inhibition of P2X7R or p38. (a) Mechanical threshold after inhibition of P2X7R. (b) Mechanical threshold after inhibition of p38. Mean ± SEM ( n = 5). ∗∗∗ P < 0.001 (vs. control); # P < 0.05; ## P < 0.01 (vs. BTZ group).
    Figure Legend Snippet: Mechanical threshold alterations after inhibition of P2X7R or p38. (a) Mechanical threshold after inhibition of P2X7R. (b) Mechanical threshold after inhibition of p38. Mean ± SEM ( n = 5). ∗∗∗ P < 0.001 (vs. control); # P < 0.05; ## P < 0.01 (vs. BTZ group).

    Techniques Used: Inhibition

    rabbit anti p2x7r monoclonal igg  (Alomone Labs)


    Bioz Verified Symbol Alomone Labs is a verified supplier
    Bioz Manufacturer Symbol Alomone Labs manufactures this product  
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  • 94

    Structured Review

    Alomone Labs rabbit anti p2x7r monoclonal igg
    The sequences of oligonucleotide primers.
    Rabbit Anti P2x7r Monoclonal Igg, 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 p2x7r monoclonal igg/product/Alomone Labs
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rabbit anti p2x7r monoclonal igg - by Bioz Stars, 2023-02
    94/100 stars

    Images

    1) Product Images from "The Actions and Mechanisms of P2X7R and p38 MAPK Activation in Mediating Bortezomib-Induced Neuropathic Pain"

    Article Title: The Actions and Mechanisms of P2X7R and p38 MAPK Activation in Mediating Bortezomib-Induced Neuropathic Pain

    Journal: BioMed Research International

    doi: 10.1155/2020/8143754

    The sequences of oligonucleotide primers.
    Figure Legend Snippet: The sequences of oligonucleotide primers.

    Techniques Used:

    The antibodies for immunoblotting.
    Figure Legend Snippet: The antibodies for immunoblotting.

    Techniques Used: Western Blot, Concentration Assay

    The antibodies for fluorescence labeling.
    Figure Legend Snippet: The antibodies for fluorescence labeling.

    Techniques Used: Fluorescence, Labeling, Concentration Assay

    Mechanical threshold and P2X7R and p-p38 expression. (a) Mechanical threshold after BTZ injection. (b, c) Western blot for P2X7R expression after BTZ treatment. (d) Immunofluorescence location of P2X7R in DRG. The arrows indicate the typical single- or double-labeled DRG neurons and satellite cells. P2X7R is not expressed in NF-200-positive neurons. P2X7R is expressed in GFAP-labeled satellite glial cells (SGCs). (e) Immunofluorescence location of p-p38 in DRG. The arrows indicate the typical single- or double-labeled DRG neurons and satellite cells. p-p38 is expressed in both MAP2-labeled neurons and GFAP-labeled SGCs. (f) Immunofluorescence location of P2X7R in SDH. The arrows indicate the typical single-labeled and double-labeled cells in SDH. P2X7R is expressed mainly in Iba-1-labeled microglial cells rather than in GFAP-labeled astrocytes and MAP2-labeled neurons. (g) Immunofluorescence location of p-p38 in SDH. The arrows indicate the typical single-labeled and double-labeled cells in SDH. p-p38 is expressed mainly in Iba-1-labeled microglial cells. Scale bar = 50 μ m. Mean ± SEM ( n = 5). ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001.
    Figure Legend Snippet: Mechanical threshold and P2X7R and p-p38 expression. (a) Mechanical threshold after BTZ injection. (b, c) Western blot for P2X7R expression after BTZ treatment. (d) Immunofluorescence location of P2X7R in DRG. The arrows indicate the typical single- or double-labeled DRG neurons and satellite cells. P2X7R is not expressed in NF-200-positive neurons. P2X7R is expressed in GFAP-labeled satellite glial cells (SGCs). (e) Immunofluorescence location of p-p38 in DRG. The arrows indicate the typical single- or double-labeled DRG neurons and satellite cells. p-p38 is expressed in both MAP2-labeled neurons and GFAP-labeled SGCs. (f) Immunofluorescence location of P2X7R in SDH. The arrows indicate the typical single-labeled and double-labeled cells in SDH. P2X7R is expressed mainly in Iba-1-labeled microglial cells rather than in GFAP-labeled astrocytes and MAP2-labeled neurons. (g) Immunofluorescence location of p-p38 in SDH. The arrows indicate the typical single-labeled and double-labeled cells in SDH. p-p38 is expressed mainly in Iba-1-labeled microglial cells. Scale bar = 50 μ m. Mean ± SEM ( n = 5). ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001.

    Techniques Used: Expressing, Injection, Western Blot, Immunofluorescence, Labeling

    p38 mRNA expression and p38 phosphorylation in DRG after inhibition of P2X7R with BBG. (a) p38 mRNA levels. (b) p-p38 protein immunoblotting bands. (c) p-p38 protein levels. (d) p-p38 immunofluorescence labeling. The arrows show the typical p-p38 single-labeled DRG cells. (e) p-p38 fluorescence density. Scale bar = 50 μ m. Mean ± SEM ( n = 5). ∗ P < 0.05; ∗∗∗ P < 0.001.
    Figure Legend Snippet: p38 mRNA expression and p38 phosphorylation in DRG after inhibition of P2X7R with BBG. (a) p38 mRNA levels. (b) p-p38 protein immunoblotting bands. (c) p-p38 protein levels. (d) p-p38 immunofluorescence labeling. The arrows show the typical p-p38 single-labeled DRG cells. (e) p-p38 fluorescence density. Scale bar = 50 μ m. Mean ± SEM ( n = 5). ∗ P < 0.05; ∗∗∗ P < 0.001.

    Techniques Used: Expressing, Inhibition, Western Blot, Immunofluorescence, Labeling, Fluorescence

    p38 mRNA expression and p38 phosphorylation in SDH after inhibition of P2X7R with BBG. (a) p38 mRNA levels. (b) p-p38 protein immunoblotting bands. (c) p-p38 protein levels. (d) P2X7R and p-p38 coexpression fluorescence labeling. The arrows indicate the typical single-labeled and double-labeled SDH microglia. (e) P2X7R and p-p38 coexpression fluorescence density. Scale bar = 50 μ m. Mean ± SEM ( n = 5). ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001.
    Figure Legend Snippet: p38 mRNA expression and p38 phosphorylation in SDH after inhibition of P2X7R with BBG. (a) p38 mRNA levels. (b) p-p38 protein immunoblotting bands. (c) p-p38 protein levels. (d) P2X7R and p-p38 coexpression fluorescence labeling. The arrows indicate the typical single-labeled and double-labeled SDH microglia. (e) P2X7R and p-p38 coexpression fluorescence density. Scale bar = 50 μ m. Mean ± SEM ( n = 5). ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001.

    Techniques Used: Expressing, Inhibition, Western Blot, Fluorescence, Labeling

    IL-1 β , IL-6, and TNF- α mRNA expression in DRG and SDH after inhibition of P2X7R. (a) DRG IL-1 β mRNA. (b) DRG IL-6 mRNA. (c) DRG TNF- α mRNA. (d) SDH IL-1 β mRNA. (e) SDH IL-6 mRNA. (f) SDH TNF- α mRNA. Mean ± SEM ( n = 5). ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001.
    Figure Legend Snippet: IL-1 β , IL-6, and TNF- α mRNA expression in DRG and SDH after inhibition of P2X7R. (a) DRG IL-1 β mRNA. (b) DRG IL-6 mRNA. (c) DRG TNF- α mRNA. (d) SDH IL-1 β mRNA. (e) SDH IL-6 mRNA. (f) SDH TNF- α mRNA. Mean ± SEM ( n = 5). ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001.

    Techniques Used: Expressing, Inhibition

    P2X7R mRNA and protein expression in DRG after inhibition of p38 phosphorylation. (a) P2X7R mRNA levels. (b) P2X7R protein immunoblotting bands. (c) P2X7R protein levels. (d) P2X7R and GFAP coexpression fluorescence labeling for SGCs. The arrows indicate the typical single-labeled and double-labeled DRG satellite cells. (e) P2X7R and GFAP coexpression fluorescence density. Scale bar = 50 μ m. Mean ± SEM ( n = 5). ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001.
    Figure Legend Snippet: P2X7R mRNA and protein expression in DRG after inhibition of p38 phosphorylation. (a) P2X7R mRNA levels. (b) P2X7R protein immunoblotting bands. (c) P2X7R protein levels. (d) P2X7R and GFAP coexpression fluorescence labeling for SGCs. The arrows indicate the typical single-labeled and double-labeled DRG satellite cells. (e) P2X7R and GFAP coexpression fluorescence density. Scale bar = 50 μ m. Mean ± SEM ( n = 5). ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001.

    Techniques Used: Expressing, Inhibition, Western Blot, Fluorescence, Labeling

    P2X7R mRNA and protein expression in SDH after inhibition of p38 phosphorylation. (a) P2X7R mRNA levels. (b) P2X7R protein immunoblotting bands. (c) P2X7R protein levels. (d) P2X7R and p-p38 coexpression fluorescence labeling. The arrows indicate the typical single-labeled and double-labeled SDH microglia. (e) P2X7R and p-p38 colocalization fluorescence density. Scale bar = 50 μ m. Mean ± SEM ( n = 5). ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001.
    Figure Legend Snippet: P2X7R mRNA and protein expression in SDH after inhibition of p38 phosphorylation. (a) P2X7R mRNA levels. (b) P2X7R protein immunoblotting bands. (c) P2X7R protein levels. (d) P2X7R and p-p38 coexpression fluorescence labeling. The arrows indicate the typical single-labeled and double-labeled SDH microglia. (e) P2X7R and p-p38 colocalization fluorescence density. Scale bar = 50 μ m. Mean ± SEM ( n = 5). ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001.

    Techniques Used: Expressing, Inhibition, Western Blot, Fluorescence, Labeling

    Mechanical threshold alterations after inhibition of P2X7R or p38. (a) Mechanical threshold after inhibition of P2X7R. (b) Mechanical threshold after inhibition of p38. Mean ± SEM ( n = 5). ∗∗∗ P < 0.001 (vs. control); # P < 0.05; ## P < 0.01 (vs. BTZ group).
    Figure Legend Snippet: Mechanical threshold alterations after inhibition of P2X7R or p38. (a) Mechanical threshold after inhibition of P2X7R. (b) Mechanical threshold after inhibition of p38. Mean ± SEM ( n = 5). ∗∗∗ P < 0.001 (vs. control); # P < 0.05; ## P < 0.01 (vs. BTZ group).

    Techniques Used: Inhibition

    anti p2x7 receptor extracellular fitc antibody  (Alomone Labs)


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    Alomone Labs anti p2x7 receptor extracellular fitc antibody
    <t>P2X7R</t> regulates the development of acute gouty arthritis in rats. ( A ) At 12h, clinical manifestations of right ankle joint in rats among ATP group, BBG group and control group. ( B and C ) Clinical score and swelling index of three groups of rats at each time point. ( D ) Inflammatory cells infiltrated the synovial tissue of the right ankle joint in three groups of rats, at 24h. ( E and F ) Infiltration of mononuclear cells and neutrophils in the synovial tissue of the ankle joint among the three groups. * P<0.05, **P< 0.01, ***P< 0.001.
    Anti P2x7 Receptor Extracellular Fitc 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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    1) Product Images from "ATP-Activated P2X7R Promote the Attack of Acute Gouty Arthritis in Rats Through Activating NLRP3 Inflammasome and Inflammatory Cytokine Production"

    Article Title: ATP-Activated P2X7R Promote the Attack of Acute Gouty Arthritis in Rats Through Activating NLRP3 Inflammasome and Inflammatory Cytokine Production

    Journal: Journal of Inflammation Research

    doi: 10.2147/JIR.S351660

    P2X7R regulates the development of acute gouty arthritis in rats. ( A ) At 12h, clinical manifestations of right ankle joint in rats among ATP group, BBG group and control group. ( B and C ) Clinical score and swelling index of three groups of rats at each time point. ( D ) Inflammatory cells infiltrated the synovial tissue of the right ankle joint in three groups of rats, at 24h. ( E and F ) Infiltration of mononuclear cells and neutrophils in the synovial tissue of the ankle joint among the three groups. * P<0.05, **P< 0.01, ***P< 0.001.
    Figure Legend Snippet: P2X7R regulates the development of acute gouty arthritis in rats. ( A ) At 12h, clinical manifestations of right ankle joint in rats among ATP group, BBG group and control group. ( B and C ) Clinical score and swelling index of three groups of rats at each time point. ( D ) Inflammatory cells infiltrated the synovial tissue of the right ankle joint in three groups of rats, at 24h. ( E and F ) Infiltration of mononuclear cells and neutrophils in the synovial tissue of the ankle joint among the three groups. * P<0.05, **P< 0.01, ***P< 0.001.

    Techniques Used:

    The expression changes of P2X7R on macrophages affect their ability to take up YO-PRO-1. ( A ) At 12h, the expression level of P2X7R in each group of macrophages was detected by flow cytometry. ( B ) Expression of P2X7R among the three groups at different time points. ( C ) At 12h, the percentage of YO-PRO-1 uptake by macrophages in each group was detected by flow cytometry. ( D ) The ability of macrophages to uptake YO-PRO-1 among the three groups at each time point. ( E ) The expression level of P2X7R was positively correlated with the ability of macrophages to uptake YO-PRO-1. * P<0.05, ** P< 0.01, *** P< 0.001.
    Figure Legend Snippet: The expression changes of P2X7R on macrophages affect their ability to take up YO-PRO-1. ( A ) At 12h, the expression level of P2X7R in each group of macrophages was detected by flow cytometry. ( B ) Expression of P2X7R among the three groups at different time points. ( C ) At 12h, the percentage of YO-PRO-1 uptake by macrophages in each group was detected by flow cytometry. ( D ) The ability of macrophages to uptake YO-PRO-1 among the three groups at each time point. ( E ) The expression level of P2X7R was positively correlated with the ability of macrophages to uptake YO-PRO-1. * P<0.05, ** P< 0.01, *** P< 0.001.

    Techniques Used: Expressing, Flow Cytometry

    P2X7R mediated NLRP3 inflammatory-dependent IL-1β secretion on macrophages. The expressions of P2X7R ( A ), NLRP3 ( B ) and IL-1β ( C ) in rat spleen macrophages were analyzed by qRT-PCR. *P<0.05, **P< 0.01, ***P< 0.001.
    Figure Legend Snippet: P2X7R mediated NLRP3 inflammatory-dependent IL-1β secretion on macrophages. The expressions of P2X7R ( A ), NLRP3 ( B ) and IL-1β ( C ) in rat spleen macrophages were analyzed by qRT-PCR. *P<0.05, **P< 0.01, ***P< 0.001.

    Techniques Used: Quantitative RT-PCR

    P2X7R promote Th17 cells and pro-inflammatory cytokines production. ( A ) The expressions of CD4 + IL-17 + Th17 cells among the three groups at 12h. ( B ) The levels of CD4 + IL-17 + Th17 cells in ATP group, BBG group and Control group at each time point. ( C-E ) The serum levels of IL-1β, IL-6 and IL-17 of the three groups at each time point. (F) The expression of IL-17 mRNA among three groups at each time point. * P<0.05, ** P< 0.01, *** P< 0.001.
    Figure Legend Snippet: P2X7R promote Th17 cells and pro-inflammatory cytokines production. ( A ) The expressions of CD4 + IL-17 + Th17 cells among the three groups at 12h. ( B ) The levels of CD4 + IL-17 + Th17 cells in ATP group, BBG group and Control group at each time point. ( C-E ) The serum levels of IL-1β, IL-6 and IL-17 of the three groups at each time point. (F) The expression of IL-17 mRNA among three groups at each time point. * P<0.05, ** P< 0.01, *** P< 0.001.

    Techniques Used: Expressing

    Tregs and anti-inflammatory cytokines in P2X7R-regulated acute gouty arthritis. ( A ) The expressions of Tregs among the three groups at 12h. ( B ) The levels of CD4 + CD25 + FOXP3 + Tregs in three groups at each time point. ( C) The expression of FOXP3 mRNA among three groups at each time point. ( D -E) The expressions of TGF-β 1 and IL-10 in serum among the three groups. *P<0.05, **P< 0.01, ***P< 0.001.
    Figure Legend Snippet: Tregs and anti-inflammatory cytokines in P2X7R-regulated acute gouty arthritis. ( A ) The expressions of Tregs among the three groups at 12h. ( B ) The levels of CD4 + CD25 + FOXP3 + Tregs in three groups at each time point. ( C) The expression of FOXP3 mRNA among three groups at each time point. ( D -E) The expressions of TGF-β 1 and IL-10 in serum among the three groups. *P<0.05, **P< 0.01, ***P< 0.001.

    Techniques Used: Expressing

    The changing trend of cytokines and Treg/Th17 cells matched with the pathogenesis process of ATP-activated P2X7R-regulated acute gouty arthritis. ( A ) The trends of P2X7R and IL-1β in ATP group. ( B ) The trends of CD4 + IL-17 + Th17 cells and IL-17 in ATP group. ( C ) The trends of CD4 + CD25 + Foxp3 + Treg cells and TGF-β 1 in ATP group. ( D ) The trends of CD4 + CD25 + Foxp3 + Treg cells and CD4 + IL-17 + Th17 cells in ATP group. ( E ) The ratios of Treg/Th17 among ATP group, BBG group and Control group. * P<0.05, ATP group vs BBG group, # P<0.05, ATP group vs Control group.
    Figure Legend Snippet: The changing trend of cytokines and Treg/Th17 cells matched with the pathogenesis process of ATP-activated P2X7R-regulated acute gouty arthritis. ( A ) The trends of P2X7R and IL-1β in ATP group. ( B ) The trends of CD4 + IL-17 + Th17 cells and IL-17 in ATP group. ( C ) The trends of CD4 + CD25 + Foxp3 + Treg cells and TGF-β 1 in ATP group. ( D ) The trends of CD4 + CD25 + Foxp3 + Treg cells and CD4 + IL-17 + Th17 cells in ATP group. ( E ) The ratios of Treg/Th17 among ATP group, BBG group and Control group. * P<0.05, ATP group vs BBG group, # P<0.05, ATP group vs Control group.

    Techniques Used:

    antibodies anti p2x7 fitc  (Alomone Labs)


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    Alomone Labs antibodies anti p2x7 fitc
    <t>P2X7</t> receptors identified by immunofluorescence, in renal cortex and medulla in Sham and Ang II-infused rats. The expression of P2X7 receptors in the Ang II group is localized in tubular membranes ( A ). Western blot ( B ) showed abundance of the P2X7 receptor protein in the Ang II group ( n = 7 per group). * p < 0.01 vs. Sham. Ang II = angiotensin II, BBG = Brilliant blue G.
    Antibodies Anti P2x7 Fitc, 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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    1) Product Images from "The Role of P2X7 Purinergic Receptors in the Renal Inflammation Associated with Angiotensin II-Induced Hypertension"

    Article Title: The Role of P2X7 Purinergic Receptors in the Renal Inflammation Associated with Angiotensin II-Induced Hypertension

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms21114041

    P2X7 receptors identified by immunofluorescence, in renal cortex and medulla in Sham and Ang II-infused rats. The expression of P2X7 receptors in the Ang II group is localized in tubular membranes ( A ). Western blot ( B ) showed abundance of the P2X7 receptor protein in the Ang II group ( n = 7 per group). * p < 0.01 vs. Sham. Ang II = angiotensin II, BBG = Brilliant blue G.
    Figure Legend Snippet: P2X7 receptors identified by immunofluorescence, in renal cortex and medulla in Sham and Ang II-infused rats. The expression of P2X7 receptors in the Ang II group is localized in tubular membranes ( A ). Western blot ( B ) showed abundance of the P2X7 receptor protein in the Ang II group ( n = 7 per group). * p < 0.01 vs. Sham. Ang II = angiotensin II, BBG = Brilliant blue G.

    Techniques Used: Immunofluorescence, Expressing, Western Blot

    P2X7 receptors (second column) in the infiltrating T cells of Ang II group co-localize with cluster differentiation (CD, first column) antigens in T cells (CD 3 positive cells), B cells (CD5 and CD20 positive cells), macrophages (CD68 positive cells) and leukocyte common antigen CD45, predominantly expressed in T lymphocytes.
    Figure Legend Snippet: P2X7 receptors (second column) in the infiltrating T cells of Ang II group co-localize with cluster differentiation (CD, first column) antigens in T cells (CD 3 positive cells), B cells (CD5 and CD20 positive cells), macrophages (CD68 positive cells) and leukocyte common antigen CD45, predominantly expressed in T lymphocytes.

    Techniques Used:

    fluorescein isothiocyanate fitc  (Alomone Labs)


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    Alomone Labs fluorescein isothiocyanate fitc
    Fluorescein Isothiocyanate Fitc, 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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    p2x7r antibody incubation  (Alomone Labs)


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    Alomone Labs p2x7r antibody incubation
    P2x7r Antibody Incubation, 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/p2x7r antibody incubation/product/Alomone Labs
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    anti p2x7r extracellular fitc  (Alomone Labs)


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    Alomone Labs anti p2x7r extracellular fitc
    P24XR and <t>P2X7R</t> Mediate ATP-Dependent Calcium Signal Propagation (A and B) Murine RAW 264.7 macrophages were loaded with photoactivable caged-IP 3 and the fluorescent calcium indicator Fluo-4 (green), and calcium signal propagation after IP 3 uncaging in the origin cell (white box) was monitored in live imaging. Experiments were performed in HBSS with 2 mM Ca 2+ (A) or in calcium-free HBSS supplemented with 2 mM EGTA (B). Scale bars, 50 μm. See also . (C and D) Quantification of 3 independent live calcium imaging experiments with RAW 264.7 (C) or primary BMDMs (D). Error bars represent SEM. For statistical data analysis, Student’s t test was used ( ∗ p < 0.05). (E and F) Relative mRNA expression of different members of the P2X family of receptors, measured by real-time PCR in the RAW 264.7 cell line (E) and in BMDM primary macrophages (F). Error bars represent SEM. (G) Quantification of 5 independent live calcium imaging experiments with RAW 264.7 cells pre-treated with the P2X4R inhibitor 5BDBD (100 μM, 30 min at 37°C), the P2X7R inhibitor A740003 (100 μM, 30 min at 37°C), or their vehicle (DMSO) or left untreated. Error bars represent SEM. For statistical data analysis, One-way ANOVA followed by Bonferroni’s multiple comparisons test was used ( ∗∗ p < 0.01; ns, non-significant). (H) Representative western blot (left) and quantification of repeated experiments (right) of P2X4R and P2X7R expression in RAW 264.7 cells transfected with siRNA specific for P2X4R and P2X7R or with scramble siRNA. Control cells were electroporated in the absence of oligonucleotides. (I) Quantification of 3 independent live calcium imaging experiments with RAW 264.7 cells silenced for P2X4R and P2X7R. Error bars represent SEM. For statistical data analysis, one-way ANOVA followed by Bonferroni’s multiple comparisons test was used ( ∗∗ p < 0.01; ns, non-significant).
    Anti P2x7r Extracellular Fitc, 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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    Images

    1) Product Images from "Intercellular Calcium Signaling Induced by ATP Potentiates Macrophage Phagocytosis"

    Article Title: Intercellular Calcium Signaling Induced by ATP Potentiates Macrophage Phagocytosis

    Journal: Cell Reports

    doi: 10.1016/j.celrep.2019.03.011

    P24XR and P2X7R Mediate ATP-Dependent Calcium Signal Propagation (A and B) Murine RAW 264.7 macrophages were loaded with photoactivable caged-IP 3 and the fluorescent calcium indicator Fluo-4 (green), and calcium signal propagation after IP 3 uncaging in the origin cell (white box) was monitored in live imaging. Experiments were performed in HBSS with 2 mM Ca 2+ (A) or in calcium-free HBSS supplemented with 2 mM EGTA (B). Scale bars, 50 μm. See also . (C and D) Quantification of 3 independent live calcium imaging experiments with RAW 264.7 (C) or primary BMDMs (D). Error bars represent SEM. For statistical data analysis, Student’s t test was used ( ∗ p < 0.05). (E and F) Relative mRNA expression of different members of the P2X family of receptors, measured by real-time PCR in the RAW 264.7 cell line (E) and in BMDM primary macrophages (F). Error bars represent SEM. (G) Quantification of 5 independent live calcium imaging experiments with RAW 264.7 cells pre-treated with the P2X4R inhibitor 5BDBD (100 μM, 30 min at 37°C), the P2X7R inhibitor A740003 (100 μM, 30 min at 37°C), or their vehicle (DMSO) or left untreated. Error bars represent SEM. For statistical data analysis, One-way ANOVA followed by Bonferroni’s multiple comparisons test was used ( ∗∗ p < 0.01; ns, non-significant). (H) Representative western blot (left) and quantification of repeated experiments (right) of P2X4R and P2X7R expression in RAW 264.7 cells transfected with siRNA specific for P2X4R and P2X7R or with scramble siRNA. Control cells were electroporated in the absence of oligonucleotides. (I) Quantification of 3 independent live calcium imaging experiments with RAW 264.7 cells silenced for P2X4R and P2X7R. Error bars represent SEM. For statistical data analysis, one-way ANOVA followed by Bonferroni’s multiple comparisons test was used ( ∗∗ p < 0.01; ns, non-significant).
    Figure Legend Snippet: P24XR and P2X7R Mediate ATP-Dependent Calcium Signal Propagation (A and B) Murine RAW 264.7 macrophages were loaded with photoactivable caged-IP 3 and the fluorescent calcium indicator Fluo-4 (green), and calcium signal propagation after IP 3 uncaging in the origin cell (white box) was monitored in live imaging. Experiments were performed in HBSS with 2 mM Ca 2+ (A) or in calcium-free HBSS supplemented with 2 mM EGTA (B). Scale bars, 50 μm. See also . (C and D) Quantification of 3 independent live calcium imaging experiments with RAW 264.7 (C) or primary BMDMs (D). Error bars represent SEM. For statistical data analysis, Student’s t test was used ( ∗ p < 0.05). (E and F) Relative mRNA expression of different members of the P2X family of receptors, measured by real-time PCR in the RAW 264.7 cell line (E) and in BMDM primary macrophages (F). Error bars represent SEM. (G) Quantification of 5 independent live calcium imaging experiments with RAW 264.7 cells pre-treated with the P2X4R inhibitor 5BDBD (100 μM, 30 min at 37°C), the P2X7R inhibitor A740003 (100 μM, 30 min at 37°C), or their vehicle (DMSO) or left untreated. Error bars represent SEM. For statistical data analysis, One-way ANOVA followed by Bonferroni’s multiple comparisons test was used ( ∗∗ p < 0.01; ns, non-significant). (H) Representative western blot (left) and quantification of repeated experiments (right) of P2X4R and P2X7R expression in RAW 264.7 cells transfected with siRNA specific for P2X4R and P2X7R or with scramble siRNA. Control cells were electroporated in the absence of oligonucleotides. (I) Quantification of 3 independent live calcium imaging experiments with RAW 264.7 cells silenced for P2X4R and P2X7R. Error bars represent SEM. For statistical data analysis, one-way ANOVA followed by Bonferroni’s multiple comparisons test was used ( ∗∗ p < 0.01; ns, non-significant).

    Techniques Used: Imaging, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Transfection

    Macrophage Polarization Status Affects Calcium Signal Propagation (A) The surface expression of P2X4R (top) and P2X7R (bottom) was analyzed by flow cytometry in resting, IFNγ-treated (10 ng/mL, 24 h), or IL4-treated (20 ng/mL, 24 h) macrophages. Histograms show the quantification 3 independent biological replicates. Error bars represent SEM. For data analysis, one-way ANOVA followed by Bonferroni’s multiple comparisons test was used (ns, non-significant; ∗∗ p < 0.01; ∗∗∗ p < 0.001). (B) Maximal back-projection of two representative live calcium imaging experiments performed with IFNγ- or IL4-treated RAW 264.7 cells loaded with caged-IP 3 and Fluo-4. The fluorescence variation 60 s after the irradiation of the origin cell (white box) is represented in false colors. (C) Representative traces of live calcium imaging experiments, showing the fluorescence variation after the uncaging of the origin cell (red) and the bystander macrophages (black). (D) Quantification of 3 independent live calcium imaging experiments. Error bars represent SEM. For data analysis, one-way ANOVA followed by Bonferroni’s multiple comparisons test was used ( ∗∗ p < 0.01; ns, non-significant).
    Figure Legend Snippet: Macrophage Polarization Status Affects Calcium Signal Propagation (A) The surface expression of P2X4R (top) and P2X7R (bottom) was analyzed by flow cytometry in resting, IFNγ-treated (10 ng/mL, 24 h), or IL4-treated (20 ng/mL, 24 h) macrophages. Histograms show the quantification 3 independent biological replicates. Error bars represent SEM. For data analysis, one-way ANOVA followed by Bonferroni’s multiple comparisons test was used (ns, non-significant; ∗∗ p < 0.01; ∗∗∗ p < 0.001). (B) Maximal back-projection of two representative live calcium imaging experiments performed with IFNγ- or IL4-treated RAW 264.7 cells loaded with caged-IP 3 and Fluo-4. The fluorescence variation 60 s after the irradiation of the origin cell (white box) is represented in false colors. (C) Representative traces of live calcium imaging experiments, showing the fluorescence variation after the uncaging of the origin cell (red) and the bystander macrophages (black). (D) Quantification of 3 independent live calcium imaging experiments. Error bars represent SEM. For data analysis, one-way ANOVA followed by Bonferroni’s multiple comparisons test was used ( ∗∗ p < 0.01; ns, non-significant).

    Techniques Used: Expressing, Flow Cytometry, Imaging, Fluorescence, Irradiation

    Extracellular ATP Is Required for Efficient Phagocytosis (A) Primary BMDMs were incubated with PhRodo E. coli fluorescent bioparticles in the presence or absence of 5 mM EGTA to chelate extracellular calcium. Phagocytosis was monitored at 15 or 30 min by flow cytometry (see <xref ref-type=Figure S4 ). Macrophages incubated with 20 μM cytochalasin D were used as negative reference. The phagocytic index was calculated as the percentage of fluorescent macrophages multiplied by their mean of fluorescence (MFI) and normalized on the cytochalasin-treated samples. (B) Primary BMDMs were loaded with the intracellular calcium chelator BAPTA-AM or its vehicle (loading solution) before performing the phagocytosis assay. (C) Primary BMDMs were incubated with PhRodo E. coli , PhRodo Zymosan, or PhRodo S. aureus fluorescent bioparticles, in the presence or absence of apyrase (5 U/mL). (D) Primary BMDMs were pretreated with the P2X4R inhibitor 5BDBD (100 μM), the P2X7R inhibitor A740003 (100 μM), or their vehicle (DMSO), or were left untreated, before performing the phagocytosis assay. (E) Phagocytosis was performed for 30 min in the presence or absence of MSC-derived EVs, pre-incubated or not with ARL-67516 (30 min, 200 μM). The graphs are representative of at least 3 independent biological replicates, each performed in technical triplicate. Error bars represent SEM. For data analysis, a two-way ANOVA followed by Tukey’s multiple comparisons test was used ( ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ns, non-significant). " title="... with the P2X4R inhibitor 5BDBD (100 μM), the P2X7R inhibitor A740003 (100 μM), or their vehicle (DMSO), ..." property="contentUrl" width="100%" height="100%"/>
    Figure Legend Snippet: Extracellular ATP Is Required for Efficient Phagocytosis (A) Primary BMDMs were incubated with PhRodo E. coli fluorescent bioparticles in the presence or absence of 5 mM EGTA to chelate extracellular calcium. Phagocytosis was monitored at 15 or 30 min by flow cytometry (see Figure S4 ). Macrophages incubated with 20 μM cytochalasin D were used as negative reference. The phagocytic index was calculated as the percentage of fluorescent macrophages multiplied by their mean of fluorescence (MFI) and normalized on the cytochalasin-treated samples. (B) Primary BMDMs were loaded with the intracellular calcium chelator BAPTA-AM or its vehicle (loading solution) before performing the phagocytosis assay. (C) Primary BMDMs were incubated with PhRodo E. coli , PhRodo Zymosan, or PhRodo S. aureus fluorescent bioparticles, in the presence or absence of apyrase (5 U/mL). (D) Primary BMDMs were pretreated with the P2X4R inhibitor 5BDBD (100 μM), the P2X7R inhibitor A740003 (100 μM), or their vehicle (DMSO), or were left untreated, before performing the phagocytosis assay. (E) Phagocytosis was performed for 30 min in the presence or absence of MSC-derived EVs, pre-incubated or not with ARL-67516 (30 min, 200 μM). The graphs are representative of at least 3 independent biological replicates, each performed in technical triplicate. Error bars represent SEM. For data analysis, a two-way ANOVA followed by Tukey’s multiple comparisons test was used ( ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ns, non-significant).

    Techniques Used: Incubation, Flow Cytometry, Fluorescence, Phagocytosis Assay, Derivative Assay


    Figure Legend Snippet:

    Techniques Used: Recombinant, Negative Control, Real-time Polymerase Chain Reaction, Software


    Figure Legend Snippet:

    Techniques Used:

    p2x 7 catalogue number apr 008 f fluorescein isothiocyanate  (Alomone Labs)


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    Alomone Labs p2x 7 catalogue number apr 008 f fluorescein isothiocyanate
    Model of pore former induced lysis. A bacterial toxin inserts a large channel or pore into the erythrocyte membrane. ATP is immediately released through the pore and activates <t>P2X</t> receptors. The membrane insertion of the toxin also causes a steep rise in the intracellular Ca 2+ concentration ([Ca 2+ ] i ), which results from Ca 2+ passing through the pore itself and from activation of P2X receptors, which are non-selective cation channels permeable to Ca 2+ . The increase in [Ca 2+ ] i activates the Ca 2+ -sensitive K + channel K Ca 3.1 and Cl − channel TMEM16A, which results in K + and Cl − efflux and cell shrinkage as obligated water follows. The cells will remain shrunken as long as the K + efflux surpasses the Na + influx via the toxin pore and the P2X receptors. Prolonged stimulation of <t>P2X</t> <t>7</t> can activate pannexins, which will contribute to the Na + influx. Eventually, the Na + influx will exceed the K + efflux and the cells will swell and eventually burst. Blockage of the P2X 1 and P2X 7 receptor has been proven as a protective measure for bacterial toxins, and for complement to carry out their toxicity. The model based on previous work [ , , , ]
    P2x 7 Catalogue Number Apr 008 F Fluorescein Isothiocyanate, 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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    1) Product Images from "Erythrocyte P2X 1 receptor expression is correlated with change in haematocrit in patients admitted to the ICU with blood pathogen-positive sepsis"

    Article Title: Erythrocyte P2X 1 receptor expression is correlated with change in haematocrit in patients admitted to the ICU with blood pathogen-positive sepsis

    Journal: Critical Care

    doi: 10.1186/s13054-018-2100-3

    Model of pore former induced lysis. A bacterial toxin inserts a large channel or pore into the erythrocyte membrane. ATP is immediately released through the pore and activates P2X receptors. The membrane insertion of the toxin also causes a steep rise in the intracellular Ca 2+ concentration ([Ca 2+ ] i ), which results from Ca 2+ passing through the pore itself and from activation of P2X receptors, which are non-selective cation channels permeable to Ca 2+ . The increase in [Ca 2+ ] i activates the Ca 2+ -sensitive K + channel K Ca 3.1 and Cl − channel TMEM16A, which results in K + and Cl − efflux and cell shrinkage as obligated water follows. The cells will remain shrunken as long as the K + efflux surpasses the Na + influx via the toxin pore and the P2X receptors. Prolonged stimulation of P2X 7 can activate pannexins, which will contribute to the Na + influx. Eventually, the Na + influx will exceed the K + efflux and the cells will swell and eventually burst. Blockage of the P2X 1 and P2X 7 receptor has been proven as a protective measure for bacterial toxins, and for complement to carry out their toxicity. The model based on previous work [ , , , ]
    Figure Legend Snippet: Model of pore former induced lysis. A bacterial toxin inserts a large channel or pore into the erythrocyte membrane. ATP is immediately released through the pore and activates P2X receptors. The membrane insertion of the toxin also causes a steep rise in the intracellular Ca 2+ concentration ([Ca 2+ ] i ), which results from Ca 2+ passing through the pore itself and from activation of P2X receptors, which are non-selective cation channels permeable to Ca 2+ . The increase in [Ca 2+ ] i activates the Ca 2+ -sensitive K + channel K Ca 3.1 and Cl − channel TMEM16A, which results in K + and Cl − efflux and cell shrinkage as obligated water follows. The cells will remain shrunken as long as the K + efflux surpasses the Na + influx via the toxin pore and the P2X receptors. Prolonged stimulation of P2X 7 can activate pannexins, which will contribute to the Na + influx. Eventually, the Na + influx will exceed the K + efflux and the cells will swell and eventually burst. Blockage of the P2X 1 and P2X 7 receptor has been proven as a protective measure for bacterial toxins, and for complement to carry out their toxicity. The model based on previous work [ , , , ]

    Techniques Used: Lysis, Concentration Assay, Activation Assay

    Freeze-thaw - control experiments. a Whole blood drawn from healthy volunteers was incubated either with vehicle (Veh), 100 μM NF449 (P2X 1 antagonist) or 100 μM A804598 (P2X 7 antagonist) and frozen and stored for 3 weeks as whole blood at − 80 °C. After thawing, lysis was measured as absorbance of free haemoglobin in supernatant at 540 nm. n = 3, mean ± SEM. b Flow cytometry of thawed whole blood samples diluted 1000-fold, to ascertain equal distribution of blood cell sub-populations. Cells were identified by forward scatter FSC and side scatter SSC and quantified based on fixed regions. c Bar graph shows data presented as mean ± SEM, n = 3. ns, not significant
    Figure Legend Snippet: Freeze-thaw - control experiments. a Whole blood drawn from healthy volunteers was incubated either with vehicle (Veh), 100 μM NF449 (P2X 1 antagonist) or 100 μM A804598 (P2X 7 antagonist) and frozen and stored for 3 weeks as whole blood at − 80 °C. After thawing, lysis was measured as absorbance of free haemoglobin in supernatant at 540 nm. n = 3, mean ± SEM. b Flow cytometry of thawed whole blood samples diluted 1000-fold, to ascertain equal distribution of blood cell sub-populations. Cells were identified by forward scatter FSC and side scatter SSC and quantified based on fixed regions. c Bar graph shows data presented as mean ± SEM, n = 3. ns, not significant

    Techniques Used: Incubation, Lysis, Flow Cytometry

    Correlation statistics
    Figure Legend Snippet: Correlation statistics

    Techniques Used: Activity Assay, Expressing

    Haematocrit and haemoglobin levels and erythrocyte P2X 7 receptor expression. a Change in haematocrit in blood pathogen-positive patients with sepsis between Emergency Department (ER) and ICU admission and P2X 7 receptor expression on erythrocytes were not correlated. The right panel shows the change in haematocrit in blood pathogen-positive patients grouped by high or low expression of P2X 7 (Δhct/hour). b Change in haematocrit in blood pathogen-negative patients with sepsis between ER and ICU admission and P2X 7 receptor expression on erythrocytes. Right panel shows the change in haematocrit in blood pathogen-negative patients grouped by high or low expression of P2X 7 (Δhct/hour). c Change in haemoglobin in blood pathogen-positive patients with sepsis between ER and ICU admission and P2X 7 receptor expression on erythrocytes were not correlated. The right panel shows change in haemoglobin in blood pathogen-positive patients grouped by high or low expression of P2X 7 (ΔHgb/hour, not significant (ns)). d Change in haemoglobin in blood pathogen-negative patients with sepsis between ER and ICU admission and P2X 7 receptor expression on erythrocytes. The right panel shows change in haemoglobin in blood pathogen-negative patients grouped by high or low expression of P2X 7 (ΔHgb/hour)
    Figure Legend Snippet: Haematocrit and haemoglobin levels and erythrocyte P2X 7 receptor expression. a Change in haematocrit in blood pathogen-positive patients with sepsis between Emergency Department (ER) and ICU admission and P2X 7 receptor expression on erythrocytes were not correlated. The right panel shows the change in haematocrit in blood pathogen-positive patients grouped by high or low expression of P2X 7 (Δhct/hour). b Change in haematocrit in blood pathogen-negative patients with sepsis between ER and ICU admission and P2X 7 receptor expression on erythrocytes. Right panel shows the change in haematocrit in blood pathogen-negative patients grouped by high or low expression of P2X 7 (Δhct/hour). c Change in haemoglobin in blood pathogen-positive patients with sepsis between ER and ICU admission and P2X 7 receptor expression on erythrocytes were not correlated. The right panel shows change in haemoglobin in blood pathogen-positive patients grouped by high or low expression of P2X 7 (ΔHgb/hour, not significant (ns)). d Change in haemoglobin in blood pathogen-negative patients with sepsis between ER and ICU admission and P2X 7 receptor expression on erythrocytes. The right panel shows change in haemoglobin in blood pathogen-negative patients grouped by high or low expression of P2X 7 (ΔHgb/hour)

    Techniques Used: Expressing

    anti mouse p2x7r  (Alomone Labs)


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    Alomone Labs anti mouse p2x7r
    (A) <t>P2X7R</t> and NLRP3 immunoprecipitation (IP) in human CD4+ T cells. Expression of NLRP3 (top blot) and P2X7R (bottom blot) is shown. Lane 1: Total protein. Lane 2: IP with NLRP3 Ab. Lane 3: IP with P2X7R Ab. Lane 4: IP with Ab alone (NLRP3 and P2X7R). Lane 5: IP with control IgG (for NLRP3 Ab in top blot, for P2X7R Ab in bottom blot). The experiment was run in triplicate (representative blot shown). (B and C) Confocal microscopy analysis (B, scale bar: 5 μm, ×100 original magnification; C, scale bars: 20 μm, ×40 original magnification) depicting baseline colocalization of P2X7R (green) and NLRP3 (red) in human CD4+ T cells. Cells were stained with DAPI (blue) and immunolabeled with anti-P2X7R (green) and anti-NLRP3 Abs (red) (n = 3). (D–F) Bar graphs depicting expression of NLRP3 mRNA by qRT-PCR (D), and protein by flow cytometry (E) and ELISA (F), evaluated in human CD4+ T cells activated with benzoyl ATP (BzATP) and treated with CE-224,535, a P2X7R inhibitor. Experiments were run in duplicate (n = 5). (G) Bar graph representing expression of NLRP3 on human CD4+P2X7R+ cells analyzed by flow cytometry upon BzATP stimulation (n = 5). (H) Representative flow dot plots of NLRP3 expression upon gating on human BzATP-stimulated CD4+P2X7R+ cells. (I) Confocal analysis (scale bar: 5 μm; ×100 original magnification) depicting colocalization of P2X7R (green) and NLRP3 (red) in CD4+ T cells upon in vitro stimulation of P2X7R with BzATP (n = 3). (J–M) Bar graphs comparing expression of NLRP3 downstream signaling Th2-related factors IL-4 (J), IRF4 (K), GATA-3 (L), and IL-10 (M) by qRT-PCR using mRNA isolated from human CD4+ T cells activated with BzATP and treated with the P2X7R inhibitor CE-224,535. Experiments were run in triplicate (n = 5). Data are expressed as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; 1-way ANOVA with Bonferroni’s post hoc test or Student’s t test. mRNA expression was normalized to β-actin (ACTB).
    Anti Mouse P2x7r, 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/anti mouse p2x7r/product/Alomone Labs
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti mouse p2x7r - by Bioz Stars, 2023-02
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    1) Product Images from "P2X7R mutation disrupts the NLRP3-mediated Th program and predicts poor cardiac allograft outcomes"

    Article Title: P2X7R mutation disrupts the NLRP3-mediated Th program and predicts poor cardiac allograft outcomes

    Journal: The Journal of Clinical Investigation

    doi: 10.1172/JCI94524

    (A) P2X7R and NLRP3 immunoprecipitation (IP) in human CD4+ T cells. Expression of NLRP3 (top blot) and P2X7R (bottom blot) is shown. Lane 1: Total protein. Lane 2: IP with NLRP3 Ab. Lane 3: IP with P2X7R Ab. Lane 4: IP with Ab alone (NLRP3 and P2X7R). Lane 5: IP with control IgG (for NLRP3 Ab in top blot, for P2X7R Ab in bottom blot). The experiment was run in triplicate (representative blot shown). (B and C) Confocal microscopy analysis (B, scale bar: 5 μm, ×100 original magnification; C, scale bars: 20 μm, ×40 original magnification) depicting baseline colocalization of P2X7R (green) and NLRP3 (red) in human CD4+ T cells. Cells were stained with DAPI (blue) and immunolabeled with anti-P2X7R (green) and anti-NLRP3 Abs (red) (n = 3). (D–F) Bar graphs depicting expression of NLRP3 mRNA by qRT-PCR (D), and protein by flow cytometry (E) and ELISA (F), evaluated in human CD4+ T cells activated with benzoyl ATP (BzATP) and treated with CE-224,535, a P2X7R inhibitor. Experiments were run in duplicate (n = 5). (G) Bar graph representing expression of NLRP3 on human CD4+P2X7R+ cells analyzed by flow cytometry upon BzATP stimulation (n = 5). (H) Representative flow dot plots of NLRP3 expression upon gating on human BzATP-stimulated CD4+P2X7R+ cells. (I) Confocal analysis (scale bar: 5 μm; ×100 original magnification) depicting colocalization of P2X7R (green) and NLRP3 (red) in CD4+ T cells upon in vitro stimulation of P2X7R with BzATP (n = 3). (J–M) Bar graphs comparing expression of NLRP3 downstream signaling Th2-related factors IL-4 (J), IRF4 (K), GATA-3 (L), and IL-10 (M) by qRT-PCR using mRNA isolated from human CD4+ T cells activated with BzATP and treated with the P2X7R inhibitor CE-224,535. Experiments were run in triplicate (n = 5). Data are expressed as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; 1-way ANOVA with Bonferroni’s post hoc test or Student’s t test. mRNA expression was normalized to β-actin (ACTB).
    Figure Legend Snippet: (A) P2X7R and NLRP3 immunoprecipitation (IP) in human CD4+ T cells. Expression of NLRP3 (top blot) and P2X7R (bottom blot) is shown. Lane 1: Total protein. Lane 2: IP with NLRP3 Ab. Lane 3: IP with P2X7R Ab. Lane 4: IP with Ab alone (NLRP3 and P2X7R). Lane 5: IP with control IgG (for NLRP3 Ab in top blot, for P2X7R Ab in bottom blot). The experiment was run in triplicate (representative blot shown). (B and C) Confocal microscopy analysis (B, scale bar: 5 μm, ×100 original magnification; C, scale bars: 20 μm, ×40 original magnification) depicting baseline colocalization of P2X7R (green) and NLRP3 (red) in human CD4+ T cells. Cells were stained with DAPI (blue) and immunolabeled with anti-P2X7R (green) and anti-NLRP3 Abs (red) (n = 3). (D–F) Bar graphs depicting expression of NLRP3 mRNA by qRT-PCR (D), and protein by flow cytometry (E) and ELISA (F), evaluated in human CD4+ T cells activated with benzoyl ATP (BzATP) and treated with CE-224,535, a P2X7R inhibitor. Experiments were run in duplicate (n = 5). (G) Bar graph representing expression of NLRP3 on human CD4+P2X7R+ cells analyzed by flow cytometry upon BzATP stimulation (n = 5). (H) Representative flow dot plots of NLRP3 expression upon gating on human BzATP-stimulated CD4+P2X7R+ cells. (I) Confocal analysis (scale bar: 5 μm; ×100 original magnification) depicting colocalization of P2X7R (green) and NLRP3 (red) in CD4+ T cells upon in vitro stimulation of P2X7R with BzATP (n = 3). (J–M) Bar graphs comparing expression of NLRP3 downstream signaling Th2-related factors IL-4 (J), IRF4 (K), GATA-3 (L), and IL-10 (M) by qRT-PCR using mRNA isolated from human CD4+ T cells activated with BzATP and treated with the P2X7R inhibitor CE-224,535. Experiments were run in triplicate (n = 5). Data are expressed as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; 1-way ANOVA with Bonferroni’s post hoc test or Student’s t test. mRNA expression was normalized to β-actin (ACTB).

    Techniques Used: Immunoprecipitation, Expressing, Confocal Microscopy, Staining, Immunolabeling, Quantitative RT-PCR, Flow Cytometry, Enzyme-linked Immunosorbent Assay, In Vitro, Isolation

    (A) A 3D representation of the full-length structure of P2X7R, highlighting the putative location of the P2X7R mutation in the C-terminal intracellular portion. (B and C) Quantification of P2X7R total protein (B, ELISA, n = 3) and of P2X7R mRNA (C, qRT-PCR, n = 10) on CD4+ T cells of carrier and noncarrier patients. Samples were run in duplicate (B) or in triplicate (C) and normalized to expression level of β-actin (ACTB). (D) Transcriptome profiling of immune-relevant genes (see also Supplemental Table 3) examined in CD4+ T cells of carrier and noncarrier cardiac-transplanted patients (n = 5). (E–G) Expression of NLRP3 mRNA using qRT-PCR (E) and NLRP3 protein using flow cytometry (F) and ELISA (G) in CD4+ T cells of carrier and noncarrier patients (n = 5). (H and I) Flow cytometric expression of NLRP3 on CD4+P2X7R+ cells of carrier patients stimulated with BzATP (n = 5). (J) Percentage of P2X7R+NLRP3+ cells of carrier and noncarrier patients analyzed by immunofluorescence (Figure 1C and Supplemental Figure 2G) (n = 3). (K) Confocal microscopy analysis (×100 original magnification) of P2X7R (green) and NLRP3 (red) coexpression in CD4+ T cells of carrier patients (n = 3). Scale bar: 5 μm. (L) Subcellular localization of NLRP3 in CD4+ T cells of carrier and of noncarrier patients (n = 3). (M and N) IL-4 (M) and IRF4 (N) gene expression detected after ChIP with NLRP3 antibody in CD4+ T cells. (n = 3). (O) Quantification of NLRP3 protein measured in CD4+ T cells treated with the ubiquitin/protease inhibitor MG132 (n = 3). Bars represent mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; Student’s t test or 2-way ANOVA with Bonferroni’s post hoc test.
    Figure Legend Snippet: (A) A 3D representation of the full-length structure of P2X7R, highlighting the putative location of the P2X7R mutation in the C-terminal intracellular portion. (B and C) Quantification of P2X7R total protein (B, ELISA, n = 3) and of P2X7R mRNA (C, qRT-PCR, n = 10) on CD4+ T cells of carrier and noncarrier patients. Samples were run in duplicate (B) or in triplicate (C) and normalized to expression level of β-actin (ACTB). (D) Transcriptome profiling of immune-relevant genes (see also Supplemental Table 3) examined in CD4+ T cells of carrier and noncarrier cardiac-transplanted patients (n = 5). (E–G) Expression of NLRP3 mRNA using qRT-PCR (E) and NLRP3 protein using flow cytometry (F) and ELISA (G) in CD4+ T cells of carrier and noncarrier patients (n = 5). (H and I) Flow cytometric expression of NLRP3 on CD4+P2X7R+ cells of carrier patients stimulated with BzATP (n = 5). (J) Percentage of P2X7R+NLRP3+ cells of carrier and noncarrier patients analyzed by immunofluorescence (Figure 1C and Supplemental Figure 2G) (n = 3). (K) Confocal microscopy analysis (×100 original magnification) of P2X7R (green) and NLRP3 (red) coexpression in CD4+ T cells of carrier patients (n = 3). Scale bar: 5 μm. (L) Subcellular localization of NLRP3 in CD4+ T cells of carrier and of noncarrier patients (n = 3). (M and N) IL-4 (M) and IRF4 (N) gene expression detected after ChIP with NLRP3 antibody in CD4+ T cells. (n = 3). (O) Quantification of NLRP3 protein measured in CD4+ T cells treated with the ubiquitin/protease inhibitor MG132 (n = 3). Bars represent mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; Student’s t test or 2-way ANOVA with Bonferroni’s post hoc test.

    Techniques Used: Mutagenesis, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Expressing, Flow Cytometry, Immunofluorescence, Confocal Microscopy, Protease Inhibitor

    (A) P2X7R–/– mice receiving bm12 heart transplantation demonstrated reduced graft survival as compared with B6 recipients (**P < 0.01), which was significantly prolonged by anti–IL-17 treatment (murine IL-17–depleting antibody) (*P < 0.05 vs. P2X7R–/–) (n = 10 mice per group). (B–D) Semiquantification of graft infiltration (B), coronary vasculopathy (C), and myocyte necrosis (D) confirmed accelerated allograft rejection in P2X7R–/– mice (n = 3). (E) Representative H&E staining (x20 original magnification) showing graft cell infiltration (top panels), vasculopathy (middle panels), and myocyte necrosis (bottom panels) in B6 and P2X7R–/– mice. Scale bars: 200 μm (middle panels), 300 μm (top and bottom panels). (F and G) Numbers of IFN-γ–producing (F) and IL-4–producing (G) cells (ELISPOT) measured in cardiac-transplanted mice (n = 3). (H–M) Percentage of CD4+IL-17+ (H), CD4+IFN-γ+ (I), CD4+IL-10+ (J), CD4+CD44hiCD62Llo (K), CD8+CD44hiCD62Llo (L), and CD4+CD25+Foxp3+ (M) cells detected by flow cytometry in B6 and P2X7R–/– cardiac-transplanted mice and in P2X7R–/– anti–IL-17–treated mice (n = 5). (N) Serum IL-17 level (Luminex) measured in B6 and P2X7R–/– cardiac-transplanted mice and in P2X7R–/– anti–IL-17–treated mice (n = 5). (O) Percentage of CD4+NLRP3+ cells analyzed by flow cytometry in P2X7R–/– and B6 mice (n = 3). (P) Number of IL-4–producing cells (ELISPOT) in P2X7R–/– and B6 mice upon allostimulation (n = 3). (Q) Serum IL-4 level (Luminex), measured in B6 and P2X7R–/– cardiac-transplanted mice (n = 5). Samples were run in duplicate (Luminex) and in triplicate (ELISPOT). Bars represent mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.001; log-rank (Mantel-Cox) test (A), Wilcoxon’s and Student’s t test (2 groups), 1-way ANOVA with Bonferroni’s post hoc test (3 groups).
    Figure Legend Snippet: (A) P2X7R–/– mice receiving bm12 heart transplantation demonstrated reduced graft survival as compared with B6 recipients (**P < 0.01), which was significantly prolonged by anti–IL-17 treatment (murine IL-17–depleting antibody) (*P < 0.05 vs. P2X7R–/–) (n = 10 mice per group). (B–D) Semiquantification of graft infiltration (B), coronary vasculopathy (C), and myocyte necrosis (D) confirmed accelerated allograft rejection in P2X7R–/– mice (n = 3). (E) Representative H&E staining (x20 original magnification) showing graft cell infiltration (top panels), vasculopathy (middle panels), and myocyte necrosis (bottom panels) in B6 and P2X7R–/– mice. Scale bars: 200 μm (middle panels), 300 μm (top and bottom panels). (F and G) Numbers of IFN-γ–producing (F) and IL-4–producing (G) cells (ELISPOT) measured in cardiac-transplanted mice (n = 3). (H–M) Percentage of CD4+IL-17+ (H), CD4+IFN-γ+ (I), CD4+IL-10+ (J), CD4+CD44hiCD62Llo (K), CD8+CD44hiCD62Llo (L), and CD4+CD25+Foxp3+ (M) cells detected by flow cytometry in B6 and P2X7R–/– cardiac-transplanted mice and in P2X7R–/– anti–IL-17–treated mice (n = 5). (N) Serum IL-17 level (Luminex) measured in B6 and P2X7R–/– cardiac-transplanted mice and in P2X7R–/– anti–IL-17–treated mice (n = 5). (O) Percentage of CD4+NLRP3+ cells analyzed by flow cytometry in P2X7R–/– and B6 mice (n = 3). (P) Number of IL-4–producing cells (ELISPOT) in P2X7R–/– and B6 mice upon allostimulation (n = 3). (Q) Serum IL-4 level (Luminex), measured in B6 and P2X7R–/– cardiac-transplanted mice (n = 5). Samples were run in duplicate (Luminex) and in triplicate (ELISPOT). Bars represent mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.001; log-rank (Mantel-Cox) test (A), Wilcoxon’s and Student’s t test (2 groups), 1-way ANOVA with Bonferroni’s post hoc test (3 groups).

    Techniques Used: Transplantation Assay, Staining, Enzyme-linked Immunospot, Flow Cytometry, Luminex

    (A) Bar graph depicting the percentage of cardiac-transplanted patients who carry the WT or mutant P2X7R allele (n = 102) with an MIT change greater than 0.5 mm (early cardiac allograft vasculopathy, in black) at 1 year after transplantation in the CTOT-05 cohort. (B) Bar graph depicting the number of acute rejection episodes in cardiac-transplanted patients who carry the WT (black) or mutant (white) P2X7R allele (n = 181) within the first year after transplant in the NIT-Bergamo cohort. (C) Bar graph depicting the percentage of cardiac-transplanted patients who carry the WT or mutant P2X7R allele (n = 130) with major adverse cardiac events (MACEs, in black) at 10 years of follow-up in the AIRT-Bologna cohort. In A and C: black, percentage of patients who experienced the event; white, percentage who were free from events. (D) Line graph depicting the estimated odds ratio (OR) for clinical outcomes recorded in the 3 cohorts of cardiac-transplanted patients who carry the WT or mutant P2X7R allele. In the NIT-Bergamo cohort, the OR was calculated based on the requirement of medical intervention for acute rejection episodes with a frequency of greater or less than 3 episodes. *P < 0.05; **P < 0.01. Supplemental Tables 7–9 report detailed analyses. Fisher’s exact and Student’s t tests. (E and F) A stable connection between P2X7R and NLRP3 is necessary to establish a physiological NLRP3-mediated Th2 program (E), while alteration in the P2X7R intracellular domain induces NLRP3 displacement and retains NLRP3 in the cell membrane, thus preventing its nuclear activity and accelerating ubiquitination of NLRP3 (F). This shifts the balance of the immune response toward Th17 cells and favors the development of immune-related events, such as allograft rejection and vasculopathy. Ub, ubiquitin; eATP, extracellular ATP.
    Figure Legend Snippet: (A) Bar graph depicting the percentage of cardiac-transplanted patients who carry the WT or mutant P2X7R allele (n = 102) with an MIT change greater than 0.5 mm (early cardiac allograft vasculopathy, in black) at 1 year after transplantation in the CTOT-05 cohort. (B) Bar graph depicting the number of acute rejection episodes in cardiac-transplanted patients who carry the WT (black) or mutant (white) P2X7R allele (n = 181) within the first year after transplant in the NIT-Bergamo cohort. (C) Bar graph depicting the percentage of cardiac-transplanted patients who carry the WT or mutant P2X7R allele (n = 130) with major adverse cardiac events (MACEs, in black) at 10 years of follow-up in the AIRT-Bologna cohort. In A and C: black, percentage of patients who experienced the event; white, percentage who were free from events. (D) Line graph depicting the estimated odds ratio (OR) for clinical outcomes recorded in the 3 cohorts of cardiac-transplanted patients who carry the WT or mutant P2X7R allele. In the NIT-Bergamo cohort, the OR was calculated based on the requirement of medical intervention for acute rejection episodes with a frequency of greater or less than 3 episodes. *P < 0.05; **P < 0.01. Supplemental Tables 7–9 report detailed analyses. Fisher’s exact and Student’s t tests. (E and F) A stable connection between P2X7R and NLRP3 is necessary to establish a physiological NLRP3-mediated Th2 program (E), while alteration in the P2X7R intracellular domain induces NLRP3 displacement and retains NLRP3 in the cell membrane, thus preventing its nuclear activity and accelerating ubiquitination of NLRP3 (F). This shifts the balance of the immune response toward Th17 cells and favors the development of immune-related events, such as allograft rejection and vasculopathy. Ub, ubiquitin; eATP, extracellular ATP.

    Techniques Used: Mutagenesis, Transplantation Assay, Activity Assay

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  • 94
    Alomone Labs anti purinergic p2x 7 r antibody
    (A). Astrocytes were treated with PACAP (20 ng/ml), or indirectly co-cultured with hMSCs in the absence and presence of PACAP (20 ng/ml) for 48 hours. The cultures were subjected to Q-PCR for measurement of the mRNA levels of GLT-1 and GLAST. Data are presented as mean ± SEM and expressed as the ratio of GLT-1 (GLAST) mRNA levels compared to control. * p <0.05 versus control. (B). Astrocytes were indirectly co-cultured with hMSCs in the absence or presence of PACAP for 48 hours. Membrane proteins were then extracted for western blotting using anti-GLT-1 antibody. The same blot was reprobed with <t>anti-P2X</t> 7 R antibody. <t>P2X</t> <t>7</t> <t>R</t> levels are presented as a loading control. Relative intensity of GLT-1 levels normalized to P2X 7 R was measured. Data are presented as mean ± SEM and expressed as a percentage of GLT-1 levels in the group with combinatorial treatment compared to that detected in the group co-cultured only with hMSCs. * p <0.05 versus the group only co-cultured with hMSCs. (C). Astrocytes were infected by lentivirus carrying lenti-GLT-1-GFP, and then co-cultured with hMSCs in the absence or presence of PACAP. Strong punctate fluorescence spots (arrowheads) were observed in the processes of astrocytes co-cultured with hMSCs in the presence of PACAP, when compared to that seen in astrocytes co-cultured only with hMSCs (arrows). Scale bar, 50 µm. (D). Astrocytes were treated with PACAP, or co-cultured with hMSCs in the absence or presence of PACAP. After 48 or 72 hours, the cultures were subjected to [ 3 H]-L-glutamate uptake analysis as described in . Data are presented as mean ± SEM and expressed as a ratio of the glutamate uptake in each treated group compared to that detected in the control group. * p <0.05 versus control.
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    Alomone Labs rabbit anti p2x7r monoclonal igg
    The sequences of oligonucleotide primers.
    Rabbit Anti P2x7r Monoclonal Igg, 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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    Alomone Labs anti p2x7 receptor extracellular fitc antibody
    <t>P2X7R</t> regulates the development of acute gouty arthritis in rats. ( A ) At 12h, clinical manifestations of right ankle joint in rats among ATP group, BBG group and control group. ( B and C ) Clinical score and swelling index of three groups of rats at each time point. ( D ) Inflammatory cells infiltrated the synovial tissue of the right ankle joint in three groups of rats, at 24h. ( E and F ) Infiltration of mononuclear cells and neutrophils in the synovial tissue of the ankle joint among the three groups. * P<0.05, **P< 0.01, ***P< 0.001.
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    Alomone Labs antibodies anti p2x7 fitc
    <t>P2X7</t> receptors identified by immunofluorescence, in renal cortex and medulla in Sham and Ang II-infused rats. The expression of P2X7 receptors in the Ang II group is localized in tubular membranes ( A ). Western blot ( B ) showed abundance of the P2X7 receptor protein in the Ang II group ( n = 7 per group). * p < 0.01 vs. Sham. Ang II = angiotensin II, BBG = Brilliant blue G.
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    Alomone Labs fluorescein isothiocyanate fitc
    <t>P2X7</t> receptors identified by immunofluorescence, in renal cortex and medulla in Sham and Ang II-infused rats. The expression of P2X7 receptors in the Ang II group is localized in tubular membranes ( A ). Western blot ( B ) showed abundance of the P2X7 receptor protein in the Ang II group ( n = 7 per group). * p < 0.01 vs. Sham. Ang II = angiotensin II, BBG = Brilliant blue G.
    Fluorescein Isothiocyanate Fitc, 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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    Alomone Labs p2x7r antibody incubation
    <t>P2X7</t> receptors identified by immunofluorescence, in renal cortex and medulla in Sham and Ang II-infused rats. The expression of P2X7 receptors in the Ang II group is localized in tubular membranes ( A ). Western blot ( B ) showed abundance of the P2X7 receptor protein in the Ang II group ( n = 7 per group). * p < 0.01 vs. Sham. Ang II = angiotensin II, BBG = Brilliant blue G.
    P2x7r Antibody Incubation, 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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    Alomone Labs anti p2x7r extracellular fitc
    P24XR and <t>P2X7R</t> Mediate ATP-Dependent Calcium Signal Propagation (A and B) Murine RAW 264.7 macrophages were loaded with photoactivable caged-IP 3 and the fluorescent calcium indicator Fluo-4 (green), and calcium signal propagation after IP 3 uncaging in the origin cell (white box) was monitored in live imaging. Experiments were performed in HBSS with 2 mM Ca 2+ (A) or in calcium-free HBSS supplemented with 2 mM EGTA (B). Scale bars, 50 μm. See also . (C and D) Quantification of 3 independent live calcium imaging experiments with RAW 264.7 (C) or primary BMDMs (D). Error bars represent SEM. For statistical data analysis, Student’s t test was used ( ∗ p < 0.05). (E and F) Relative mRNA expression of different members of the P2X family of receptors, measured by real-time PCR in the RAW 264.7 cell line (E) and in BMDM primary macrophages (F). Error bars represent SEM. (G) Quantification of 5 independent live calcium imaging experiments with RAW 264.7 cells pre-treated with the P2X4R inhibitor 5BDBD (100 μM, 30 min at 37°C), the P2X7R inhibitor A740003 (100 μM, 30 min at 37°C), or their vehicle (DMSO) or left untreated. Error bars represent SEM. For statistical data analysis, One-way ANOVA followed by Bonferroni’s multiple comparisons test was used ( ∗∗ p < 0.01; ns, non-significant). (H) Representative western blot (left) and quantification of repeated experiments (right) of P2X4R and P2X7R expression in RAW 264.7 cells transfected with siRNA specific for P2X4R and P2X7R or with scramble siRNA. Control cells were electroporated in the absence of oligonucleotides. (I) Quantification of 3 independent live calcium imaging experiments with RAW 264.7 cells silenced for P2X4R and P2X7R. Error bars represent SEM. For statistical data analysis, one-way ANOVA followed by Bonferroni’s multiple comparisons test was used ( ∗∗ p < 0.01; ns, non-significant).
    Anti P2x7r Extracellular Fitc, 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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    Alomone Labs p2x 7 catalogue number apr 008 f fluorescein isothiocyanate
    Model of pore former induced lysis. A bacterial toxin inserts a large channel or pore into the erythrocyte membrane. ATP is immediately released through the pore and activates <t>P2X</t> receptors. The membrane insertion of the toxin also causes a steep rise in the intracellular Ca 2+ concentration ([Ca 2+ ] i ), which results from Ca 2+ passing through the pore itself and from activation of P2X receptors, which are non-selective cation channels permeable to Ca 2+ . The increase in [Ca 2+ ] i activates the Ca 2+ -sensitive K + channel K Ca 3.1 and Cl − channel TMEM16A, which results in K + and Cl − efflux and cell shrinkage as obligated water follows. The cells will remain shrunken as long as the K + efflux surpasses the Na + influx via the toxin pore and the P2X receptors. Prolonged stimulation of <t>P2X</t> <t>7</t> can activate pannexins, which will contribute to the Na + influx. Eventually, the Na + influx will exceed the K + efflux and the cells will swell and eventually burst. Blockage of the P2X 1 and P2X 7 receptor has been proven as a protective measure for bacterial toxins, and for complement to carry out their toxicity. The model based on previous work [ , , , ]
    P2x 7 Catalogue Number Apr 008 F Fluorescein Isothiocyanate, 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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    Alomone Labs anti mouse p2x7r
    (A) <t>P2X7R</t> and NLRP3 immunoprecipitation (IP) in human CD4+ T cells. Expression of NLRP3 (top blot) and P2X7R (bottom blot) is shown. Lane 1: Total protein. Lane 2: IP with NLRP3 Ab. Lane 3: IP with P2X7R Ab. Lane 4: IP with Ab alone (NLRP3 and P2X7R). Lane 5: IP with control IgG (for NLRP3 Ab in top blot, for P2X7R Ab in bottom blot). The experiment was run in triplicate (representative blot shown). (B and C) Confocal microscopy analysis (B, scale bar: 5 μm, ×100 original magnification; C, scale bars: 20 μm, ×40 original magnification) depicting baseline colocalization of P2X7R (green) and NLRP3 (red) in human CD4+ T cells. Cells were stained with DAPI (blue) and immunolabeled with anti-P2X7R (green) and anti-NLRP3 Abs (red) (n = 3). (D–F) Bar graphs depicting expression of NLRP3 mRNA by qRT-PCR (D), and protein by flow cytometry (E) and ELISA (F), evaluated in human CD4+ T cells activated with benzoyl ATP (BzATP) and treated with CE-224,535, a P2X7R inhibitor. Experiments were run in duplicate (n = 5). (G) Bar graph representing expression of NLRP3 on human CD4+P2X7R+ cells analyzed by flow cytometry upon BzATP stimulation (n = 5). (H) Representative flow dot plots of NLRP3 expression upon gating on human BzATP-stimulated CD4+P2X7R+ cells. (I) Confocal analysis (scale bar: 5 μm; ×100 original magnification) depicting colocalization of P2X7R (green) and NLRP3 (red) in CD4+ T cells upon in vitro stimulation of P2X7R with BzATP (n = 3). (J–M) Bar graphs comparing expression of NLRP3 downstream signaling Th2-related factors IL-4 (J), IRF4 (K), GATA-3 (L), and IL-10 (M) by qRT-PCR using mRNA isolated from human CD4+ T cells activated with BzATP and treated with the P2X7R inhibitor CE-224,535. Experiments were run in triplicate (n = 5). Data are expressed as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; 1-way ANOVA with Bonferroni’s post hoc test or Student’s t test. mRNA expression was normalized to β-actin (ACTB).
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    (A). Astrocytes were treated with PACAP (20 ng/ml), or indirectly co-cultured with hMSCs in the absence and presence of PACAP (20 ng/ml) for 48 hours. The cultures were subjected to Q-PCR for measurement of the mRNA levels of GLT-1 and GLAST. Data are presented as mean ± SEM and expressed as the ratio of GLT-1 (GLAST) mRNA levels compared to control. * p <0.05 versus control. (B). Astrocytes were indirectly co-cultured with hMSCs in the absence or presence of PACAP for 48 hours. Membrane proteins were then extracted for western blotting using anti-GLT-1 antibody. The same blot was reprobed with anti-P2X 7 R antibody. P2X 7 R levels are presented as a loading control. Relative intensity of GLT-1 levels normalized to P2X 7 R was measured. Data are presented as mean ± SEM and expressed as a percentage of GLT-1 levels in the group with combinatorial treatment compared to that detected in the group co-cultured only with hMSCs. * p <0.05 versus the group only co-cultured with hMSCs. (C). Astrocytes were infected by lentivirus carrying lenti-GLT-1-GFP, and then co-cultured with hMSCs in the absence or presence of PACAP. Strong punctate fluorescence spots (arrowheads) were observed in the processes of astrocytes co-cultured with hMSCs in the presence of PACAP, when compared to that seen in astrocytes co-cultured only with hMSCs (arrows). Scale bar, 50 µm. (D). Astrocytes were treated with PACAP, or co-cultured with hMSCs in the absence or presence of PACAP. After 48 or 72 hours, the cultures were subjected to [ 3 H]-L-glutamate uptake analysis as described in . Data are presented as mean ± SEM and expressed as a ratio of the glutamate uptake in each treated group compared to that detected in the control group. * p <0.05 versus control.

    Journal: PLoS ONE

    Article Title: Effects of Combinatorial Treatment with Pituitary Adenylate Cyclase Activating Peptide and Human Mesenchymal Stem Cells on Spinal Cord Tissue Repair

    doi: 10.1371/journal.pone.0015299

    Figure Lengend Snippet: (A). Astrocytes were treated with PACAP (20 ng/ml), or indirectly co-cultured with hMSCs in the absence and presence of PACAP (20 ng/ml) for 48 hours. The cultures were subjected to Q-PCR for measurement of the mRNA levels of GLT-1 and GLAST. Data are presented as mean ± SEM and expressed as the ratio of GLT-1 (GLAST) mRNA levels compared to control. * p <0.05 versus control. (B). Astrocytes were indirectly co-cultured with hMSCs in the absence or presence of PACAP for 48 hours. Membrane proteins were then extracted for western blotting using anti-GLT-1 antibody. The same blot was reprobed with anti-P2X 7 R antibody. P2X 7 R levels are presented as a loading control. Relative intensity of GLT-1 levels normalized to P2X 7 R was measured. Data are presented as mean ± SEM and expressed as a percentage of GLT-1 levels in the group with combinatorial treatment compared to that detected in the group co-cultured only with hMSCs. * p <0.05 versus the group only co-cultured with hMSCs. (C). Astrocytes were infected by lentivirus carrying lenti-GLT-1-GFP, and then co-cultured with hMSCs in the absence or presence of PACAP. Strong punctate fluorescence spots (arrowheads) were observed in the processes of astrocytes co-cultured with hMSCs in the presence of PACAP, when compared to that seen in astrocytes co-cultured only with hMSCs (arrows). Scale bar, 50 µm. (D). Astrocytes were treated with PACAP, or co-cultured with hMSCs in the absence or presence of PACAP. After 48 or 72 hours, the cultures were subjected to [ 3 H]-L-glutamate uptake analysis as described in . Data are presented as mean ± SEM and expressed as a ratio of the glutamate uptake in each treated group compared to that detected in the control group. * p <0.05 versus control.

    Article Snippet: The protein was identified by incubating the membrane with anti-GLT-1 antibody (1∶1000; Millipore) or anti- purinergic P2X 7 R antibody (1∶1000; Alomone Labs, Israel) overnight at 4°C, followed by HRP-conjugated secondary antibody (1∶2000) and ECL solution.

    Techniques: Cell Culture, Western Blot, Infection, Fluorescence

    The sequences of oligonucleotide primers.

    Journal: BioMed Research International

    Article Title: The Actions and Mechanisms of P2X7R and p38 MAPK Activation in Mediating Bortezomib-Induced Neuropathic Pain

    doi: 10.1155/2020/8143754

    Figure Lengend Snippet: The sequences of oligonucleotide primers.

    Article Snippet: Primary , Rabbit anti-P2X7R monoclonal IgG , 1 : 400 , Alomone Labs, Jerusalem, Israel.

    Techniques:

    The antibodies for immunoblotting.

    Journal: BioMed Research International

    Article Title: The Actions and Mechanisms of P2X7R and p38 MAPK Activation in Mediating Bortezomib-Induced Neuropathic Pain

    doi: 10.1155/2020/8143754

    Figure Lengend Snippet: The antibodies for immunoblotting.

    Article Snippet: Primary , Rabbit anti-P2X7R monoclonal IgG , 1 : 400 , Alomone Labs, Jerusalem, Israel.

    Techniques: Western Blot, Concentration Assay

    The antibodies for fluorescence labeling.

    Journal: BioMed Research International

    Article Title: The Actions and Mechanisms of P2X7R and p38 MAPK Activation in Mediating Bortezomib-Induced Neuropathic Pain

    doi: 10.1155/2020/8143754

    Figure Lengend Snippet: The antibodies for fluorescence labeling.

    Article Snippet: Primary , Rabbit anti-P2X7R monoclonal IgG , 1 : 400 , Alomone Labs, Jerusalem, Israel.

    Techniques: Fluorescence, Labeling, Concentration Assay

    Mechanical threshold and P2X7R and p-p38 expression. (a) Mechanical threshold after BTZ injection. (b, c) Western blot for P2X7R expression after BTZ treatment. (d) Immunofluorescence location of P2X7R in DRG. The arrows indicate the typical single- or double-labeled DRG neurons and satellite cells. P2X7R is not expressed in NF-200-positive neurons. P2X7R is expressed in GFAP-labeled satellite glial cells (SGCs). (e) Immunofluorescence location of p-p38 in DRG. The arrows indicate the typical single- or double-labeled DRG neurons and satellite cells. p-p38 is expressed in both MAP2-labeled neurons and GFAP-labeled SGCs. (f) Immunofluorescence location of P2X7R in SDH. The arrows indicate the typical single-labeled and double-labeled cells in SDH. P2X7R is expressed mainly in Iba-1-labeled microglial cells rather than in GFAP-labeled astrocytes and MAP2-labeled neurons. (g) Immunofluorescence location of p-p38 in SDH. The arrows indicate the typical single-labeled and double-labeled cells in SDH. p-p38 is expressed mainly in Iba-1-labeled microglial cells. Scale bar = 50 μ m. Mean ± SEM ( n = 5). ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001.

    Journal: BioMed Research International

    Article Title: The Actions and Mechanisms of P2X7R and p38 MAPK Activation in Mediating Bortezomib-Induced Neuropathic Pain

    doi: 10.1155/2020/8143754

    Figure Lengend Snippet: Mechanical threshold and P2X7R and p-p38 expression. (a) Mechanical threshold after BTZ injection. (b, c) Western blot for P2X7R expression after BTZ treatment. (d) Immunofluorescence location of P2X7R in DRG. The arrows indicate the typical single- or double-labeled DRG neurons and satellite cells. P2X7R is not expressed in NF-200-positive neurons. P2X7R is expressed in GFAP-labeled satellite glial cells (SGCs). (e) Immunofluorescence location of p-p38 in DRG. The arrows indicate the typical single- or double-labeled DRG neurons and satellite cells. p-p38 is expressed in both MAP2-labeled neurons and GFAP-labeled SGCs. (f) Immunofluorescence location of P2X7R in SDH. The arrows indicate the typical single-labeled and double-labeled cells in SDH. P2X7R is expressed mainly in Iba-1-labeled microglial cells rather than in GFAP-labeled astrocytes and MAP2-labeled neurons. (g) Immunofluorescence location of p-p38 in SDH. The arrows indicate the typical single-labeled and double-labeled cells in SDH. p-p38 is expressed mainly in Iba-1-labeled microglial cells. Scale bar = 50 μ m. Mean ± SEM ( n = 5). ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001.

    Article Snippet: Primary , Rabbit anti-P2X7R monoclonal IgG , 1 : 400 , Alomone Labs, Jerusalem, Israel.

    Techniques: Expressing, Injection, Western Blot, Immunofluorescence, Labeling

    p38 mRNA expression and p38 phosphorylation in DRG after inhibition of P2X7R with BBG. (a) p38 mRNA levels. (b) p-p38 protein immunoblotting bands. (c) p-p38 protein levels. (d) p-p38 immunofluorescence labeling. The arrows show the typical p-p38 single-labeled DRG cells. (e) p-p38 fluorescence density. Scale bar = 50 μ m. Mean ± SEM ( n = 5). ∗ P < 0.05; ∗∗∗ P < 0.001.

    Journal: BioMed Research International

    Article Title: The Actions and Mechanisms of P2X7R and p38 MAPK Activation in Mediating Bortezomib-Induced Neuropathic Pain

    doi: 10.1155/2020/8143754

    Figure Lengend Snippet: p38 mRNA expression and p38 phosphorylation in DRG after inhibition of P2X7R with BBG. (a) p38 mRNA levels. (b) p-p38 protein immunoblotting bands. (c) p-p38 protein levels. (d) p-p38 immunofluorescence labeling. The arrows show the typical p-p38 single-labeled DRG cells. (e) p-p38 fluorescence density. Scale bar = 50 μ m. Mean ± SEM ( n = 5). ∗ P < 0.05; ∗∗∗ P < 0.001.

    Article Snippet: Primary , Rabbit anti-P2X7R monoclonal IgG , 1 : 400 , Alomone Labs, Jerusalem, Israel.

    Techniques: Expressing, Inhibition, Western Blot, Immunofluorescence, Labeling, Fluorescence

    p38 mRNA expression and p38 phosphorylation in SDH after inhibition of P2X7R with BBG. (a) p38 mRNA levels. (b) p-p38 protein immunoblotting bands. (c) p-p38 protein levels. (d) P2X7R and p-p38 coexpression fluorescence labeling. The arrows indicate the typical single-labeled and double-labeled SDH microglia. (e) P2X7R and p-p38 coexpression fluorescence density. Scale bar = 50 μ m. Mean ± SEM ( n = 5). ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001.

    Journal: BioMed Research International

    Article Title: The Actions and Mechanisms of P2X7R and p38 MAPK Activation in Mediating Bortezomib-Induced Neuropathic Pain

    doi: 10.1155/2020/8143754

    Figure Lengend Snippet: p38 mRNA expression and p38 phosphorylation in SDH after inhibition of P2X7R with BBG. (a) p38 mRNA levels. (b) p-p38 protein immunoblotting bands. (c) p-p38 protein levels. (d) P2X7R and p-p38 coexpression fluorescence labeling. The arrows indicate the typical single-labeled and double-labeled SDH microglia. (e) P2X7R and p-p38 coexpression fluorescence density. Scale bar = 50 μ m. Mean ± SEM ( n = 5). ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001.

    Article Snippet: Primary , Rabbit anti-P2X7R monoclonal IgG , 1 : 400 , Alomone Labs, Jerusalem, Israel.

    Techniques: Expressing, Inhibition, Western Blot, Fluorescence, Labeling

    IL-1 β , IL-6, and TNF- α mRNA expression in DRG and SDH after inhibition of P2X7R. (a) DRG IL-1 β mRNA. (b) DRG IL-6 mRNA. (c) DRG TNF- α mRNA. (d) SDH IL-1 β mRNA. (e) SDH IL-6 mRNA. (f) SDH TNF- α mRNA. Mean ± SEM ( n = 5). ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001.

    Journal: BioMed Research International

    Article Title: The Actions and Mechanisms of P2X7R and p38 MAPK Activation in Mediating Bortezomib-Induced Neuropathic Pain

    doi: 10.1155/2020/8143754

    Figure Lengend Snippet: IL-1 β , IL-6, and TNF- α mRNA expression in DRG and SDH after inhibition of P2X7R. (a) DRG IL-1 β mRNA. (b) DRG IL-6 mRNA. (c) DRG TNF- α mRNA. (d) SDH IL-1 β mRNA. (e) SDH IL-6 mRNA. (f) SDH TNF- α mRNA. Mean ± SEM ( n = 5). ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001.

    Article Snippet: Primary , Rabbit anti-P2X7R monoclonal IgG , 1 : 400 , Alomone Labs, Jerusalem, Israel.

    Techniques: Expressing, Inhibition

    P2X7R mRNA and protein expression in DRG after inhibition of p38 phosphorylation. (a) P2X7R mRNA levels. (b) P2X7R protein immunoblotting bands. (c) P2X7R protein levels. (d) P2X7R and GFAP coexpression fluorescence labeling for SGCs. The arrows indicate the typical single-labeled and double-labeled DRG satellite cells. (e) P2X7R and GFAP coexpression fluorescence density. Scale bar = 50 μ m. Mean ± SEM ( n = 5). ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001.

    Journal: BioMed Research International

    Article Title: The Actions and Mechanisms of P2X7R and p38 MAPK Activation in Mediating Bortezomib-Induced Neuropathic Pain

    doi: 10.1155/2020/8143754

    Figure Lengend Snippet: P2X7R mRNA and protein expression in DRG after inhibition of p38 phosphorylation. (a) P2X7R mRNA levels. (b) P2X7R protein immunoblotting bands. (c) P2X7R protein levels. (d) P2X7R and GFAP coexpression fluorescence labeling for SGCs. The arrows indicate the typical single-labeled and double-labeled DRG satellite cells. (e) P2X7R and GFAP coexpression fluorescence density. Scale bar = 50 μ m. Mean ± SEM ( n = 5). ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001.

    Article Snippet: Primary , Rabbit anti-P2X7R monoclonal IgG , 1 : 400 , Alomone Labs, Jerusalem, Israel.

    Techniques: Expressing, Inhibition, Western Blot, Fluorescence, Labeling

    P2X7R mRNA and protein expression in SDH after inhibition of p38 phosphorylation. (a) P2X7R mRNA levels. (b) P2X7R protein immunoblotting bands. (c) P2X7R protein levels. (d) P2X7R and p-p38 coexpression fluorescence labeling. The arrows indicate the typical single-labeled and double-labeled SDH microglia. (e) P2X7R and p-p38 colocalization fluorescence density. Scale bar = 50 μ m. Mean ± SEM ( n = 5). ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001.

    Journal: BioMed Research International

    Article Title: The Actions and Mechanisms of P2X7R and p38 MAPK Activation in Mediating Bortezomib-Induced Neuropathic Pain

    doi: 10.1155/2020/8143754

    Figure Lengend Snippet: P2X7R mRNA and protein expression in SDH after inhibition of p38 phosphorylation. (a) P2X7R mRNA levels. (b) P2X7R protein immunoblotting bands. (c) P2X7R protein levels. (d) P2X7R and p-p38 coexpression fluorescence labeling. The arrows indicate the typical single-labeled and double-labeled SDH microglia. (e) P2X7R and p-p38 colocalization fluorescence density. Scale bar = 50 μ m. Mean ± SEM ( n = 5). ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001.

    Article Snippet: Primary , Rabbit anti-P2X7R monoclonal IgG , 1 : 400 , Alomone Labs, Jerusalem, Israel.

    Techniques: Expressing, Inhibition, Western Blot, Fluorescence, Labeling

    Mechanical threshold alterations after inhibition of P2X7R or p38. (a) Mechanical threshold after inhibition of P2X7R. (b) Mechanical threshold after inhibition of p38. Mean ± SEM ( n = 5). ∗∗∗ P < 0.001 (vs. control); # P < 0.05; ## P < 0.01 (vs. BTZ group).

    Journal: BioMed Research International

    Article Title: The Actions and Mechanisms of P2X7R and p38 MAPK Activation in Mediating Bortezomib-Induced Neuropathic Pain

    doi: 10.1155/2020/8143754

    Figure Lengend Snippet: Mechanical threshold alterations after inhibition of P2X7R or p38. (a) Mechanical threshold after inhibition of P2X7R. (b) Mechanical threshold after inhibition of p38. Mean ± SEM ( n = 5). ∗∗∗ P < 0.001 (vs. control); # P < 0.05; ## P < 0.01 (vs. BTZ group).

    Article Snippet: Primary , Rabbit anti-P2X7R monoclonal IgG , 1 : 400 , Alomone Labs, Jerusalem, Israel.

    Techniques: Inhibition

    P2X7R regulates the development of acute gouty arthritis in rats. ( A ) At 12h, clinical manifestations of right ankle joint in rats among ATP group, BBG group and control group. ( B and C ) Clinical score and swelling index of three groups of rats at each time point. ( D ) Inflammatory cells infiltrated the synovial tissue of the right ankle joint in three groups of rats, at 24h. ( E and F ) Infiltration of mononuclear cells and neutrophils in the synovial tissue of the ankle joint among the three groups. * P<0.05, **P< 0.01, ***P< 0.001.

    Journal: Journal of Inflammation Research

    Article Title: ATP-Activated P2X7R Promote the Attack of Acute Gouty Arthritis in Rats Through Activating NLRP3 Inflammasome and Inflammatory Cytokine Production

    doi: 10.2147/JIR.S351660

    Figure Lengend Snippet: P2X7R regulates the development of acute gouty arthritis in rats. ( A ) At 12h, clinical manifestations of right ankle joint in rats among ATP group, BBG group and control group. ( B and C ) Clinical score and swelling index of three groups of rats at each time point. ( D ) Inflammatory cells infiltrated the synovial tissue of the right ankle joint in three groups of rats, at 24h. ( E and F ) Infiltration of mononuclear cells and neutrophils in the synovial tissue of the ankle joint among the three groups. * P<0.05, **P< 0.01, ***P< 0.001.

    Article Snippet: The treated spleen cells (1×10^6cells) were stained with anti-P2X7 receptor (extracellular)-FITC antibody (Alomone, Jerusalem, Israel) and CD68 antibody (NOVUS, Littleton, USA), and the white cell suspension obtained from the spleen was stained with FITC conjugated mouse anti-rat CD4, PE conjugated mouse anti-rat CD25 (BD Bioscience, MD, USA), PE conjugated anti-IL-17A and APC conjugated anti-Foxp3 antibodies (eBioscience, San Jose, CA, USA).

    Techniques:

    The expression changes of P2X7R on macrophages affect their ability to take up YO-PRO-1. ( A ) At 12h, the expression level of P2X7R in each group of macrophages was detected by flow cytometry. ( B ) Expression of P2X7R among the three groups at different time points. ( C ) At 12h, the percentage of YO-PRO-1 uptake by macrophages in each group was detected by flow cytometry. ( D ) The ability of macrophages to uptake YO-PRO-1 among the three groups at each time point. ( E ) The expression level of P2X7R was positively correlated with the ability of macrophages to uptake YO-PRO-1. * P<0.05, ** P< 0.01, *** P< 0.001.

    Journal: Journal of Inflammation Research

    Article Title: ATP-Activated P2X7R Promote the Attack of Acute Gouty Arthritis in Rats Through Activating NLRP3 Inflammasome and Inflammatory Cytokine Production

    doi: 10.2147/JIR.S351660

    Figure Lengend Snippet: The expression changes of P2X7R on macrophages affect their ability to take up YO-PRO-1. ( A ) At 12h, the expression level of P2X7R in each group of macrophages was detected by flow cytometry. ( B ) Expression of P2X7R among the three groups at different time points. ( C ) At 12h, the percentage of YO-PRO-1 uptake by macrophages in each group was detected by flow cytometry. ( D ) The ability of macrophages to uptake YO-PRO-1 among the three groups at each time point. ( E ) The expression level of P2X7R was positively correlated with the ability of macrophages to uptake YO-PRO-1. * P<0.05, ** P< 0.01, *** P< 0.001.

    Article Snippet: The treated spleen cells (1×10^6cells) were stained with anti-P2X7 receptor (extracellular)-FITC antibody (Alomone, Jerusalem, Israel) and CD68 antibody (NOVUS, Littleton, USA), and the white cell suspension obtained from the spleen was stained with FITC conjugated mouse anti-rat CD4, PE conjugated mouse anti-rat CD25 (BD Bioscience, MD, USA), PE conjugated anti-IL-17A and APC conjugated anti-Foxp3 antibodies (eBioscience, San Jose, CA, USA).

    Techniques: Expressing, Flow Cytometry

    P2X7R mediated NLRP3 inflammatory-dependent IL-1β secretion on macrophages. The expressions of P2X7R ( A ), NLRP3 ( B ) and IL-1β ( C ) in rat spleen macrophages were analyzed by qRT-PCR. *P<0.05, **P< 0.01, ***P< 0.001.

    Journal: Journal of Inflammation Research

    Article Title: ATP-Activated P2X7R Promote the Attack of Acute Gouty Arthritis in Rats Through Activating NLRP3 Inflammasome and Inflammatory Cytokine Production

    doi: 10.2147/JIR.S351660

    Figure Lengend Snippet: P2X7R mediated NLRP3 inflammatory-dependent IL-1β secretion on macrophages. The expressions of P2X7R ( A ), NLRP3 ( B ) and IL-1β ( C ) in rat spleen macrophages were analyzed by qRT-PCR. *P<0.05, **P< 0.01, ***P< 0.001.

    Article Snippet: The treated spleen cells (1×10^6cells) were stained with anti-P2X7 receptor (extracellular)-FITC antibody (Alomone, Jerusalem, Israel) and CD68 antibody (NOVUS, Littleton, USA), and the white cell suspension obtained from the spleen was stained with FITC conjugated mouse anti-rat CD4, PE conjugated mouse anti-rat CD25 (BD Bioscience, MD, USA), PE conjugated anti-IL-17A and APC conjugated anti-Foxp3 antibodies (eBioscience, San Jose, CA, USA).

    Techniques: Quantitative RT-PCR

    P2X7R promote Th17 cells and pro-inflammatory cytokines production. ( A ) The expressions of CD4 + IL-17 + Th17 cells among the three groups at 12h. ( B ) The levels of CD4 + IL-17 + Th17 cells in ATP group, BBG group and Control group at each time point. ( C-E ) The serum levels of IL-1β, IL-6 and IL-17 of the three groups at each time point. (F) The expression of IL-17 mRNA among three groups at each time point. * P<0.05, ** P< 0.01, *** P< 0.001.

    Journal: Journal of Inflammation Research

    Article Title: ATP-Activated P2X7R Promote the Attack of Acute Gouty Arthritis in Rats Through Activating NLRP3 Inflammasome and Inflammatory Cytokine Production

    doi: 10.2147/JIR.S351660

    Figure Lengend Snippet: P2X7R promote Th17 cells and pro-inflammatory cytokines production. ( A ) The expressions of CD4 + IL-17 + Th17 cells among the three groups at 12h. ( B ) The levels of CD4 + IL-17 + Th17 cells in ATP group, BBG group and Control group at each time point. ( C-E ) The serum levels of IL-1β, IL-6 and IL-17 of the three groups at each time point. (F) The expression of IL-17 mRNA among three groups at each time point. * P<0.05, ** P< 0.01, *** P< 0.001.

    Article Snippet: The treated spleen cells (1×10^6cells) were stained with anti-P2X7 receptor (extracellular)-FITC antibody (Alomone, Jerusalem, Israel) and CD68 antibody (NOVUS, Littleton, USA), and the white cell suspension obtained from the spleen was stained with FITC conjugated mouse anti-rat CD4, PE conjugated mouse anti-rat CD25 (BD Bioscience, MD, USA), PE conjugated anti-IL-17A and APC conjugated anti-Foxp3 antibodies (eBioscience, San Jose, CA, USA).

    Techniques: Expressing

    Tregs and anti-inflammatory cytokines in P2X7R-regulated acute gouty arthritis. ( A ) The expressions of Tregs among the three groups at 12h. ( B ) The levels of CD4 + CD25 + FOXP3 + Tregs in three groups at each time point. ( C) The expression of FOXP3 mRNA among three groups at each time point. ( D -E) The expressions of TGF-β 1 and IL-10 in serum among the three groups. *P<0.05, **P< 0.01, ***P< 0.001.

    Journal: Journal of Inflammation Research

    Article Title: ATP-Activated P2X7R Promote the Attack of Acute Gouty Arthritis in Rats Through Activating NLRP3 Inflammasome and Inflammatory Cytokine Production

    doi: 10.2147/JIR.S351660

    Figure Lengend Snippet: Tregs and anti-inflammatory cytokines in P2X7R-regulated acute gouty arthritis. ( A ) The expressions of Tregs among the three groups at 12h. ( B ) The levels of CD4 + CD25 + FOXP3 + Tregs in three groups at each time point. ( C) The expression of FOXP3 mRNA among three groups at each time point. ( D -E) The expressions of TGF-β 1 and IL-10 in serum among the three groups. *P<0.05, **P< 0.01, ***P< 0.001.

    Article Snippet: The treated spleen cells (1×10^6cells) were stained with anti-P2X7 receptor (extracellular)-FITC antibody (Alomone, Jerusalem, Israel) and CD68 antibody (NOVUS, Littleton, USA), and the white cell suspension obtained from the spleen was stained with FITC conjugated mouse anti-rat CD4, PE conjugated mouse anti-rat CD25 (BD Bioscience, MD, USA), PE conjugated anti-IL-17A and APC conjugated anti-Foxp3 antibodies (eBioscience, San Jose, CA, USA).

    Techniques: Expressing

    The changing trend of cytokines and Treg/Th17 cells matched with the pathogenesis process of ATP-activated P2X7R-regulated acute gouty arthritis. ( A ) The trends of P2X7R and IL-1β in ATP group. ( B ) The trends of CD4 + IL-17 + Th17 cells and IL-17 in ATP group. ( C ) The trends of CD4 + CD25 + Foxp3 + Treg cells and TGF-β 1 in ATP group. ( D ) The trends of CD4 + CD25 + Foxp3 + Treg cells and CD4 + IL-17 + Th17 cells in ATP group. ( E ) The ratios of Treg/Th17 among ATP group, BBG group and Control group. * P<0.05, ATP group vs BBG group, # P<0.05, ATP group vs Control group.

    Journal: Journal of Inflammation Research

    Article Title: ATP-Activated P2X7R Promote the Attack of Acute Gouty Arthritis in Rats Through Activating NLRP3 Inflammasome and Inflammatory Cytokine Production

    doi: 10.2147/JIR.S351660

    Figure Lengend Snippet: The changing trend of cytokines and Treg/Th17 cells matched with the pathogenesis process of ATP-activated P2X7R-regulated acute gouty arthritis. ( A ) The trends of P2X7R and IL-1β in ATP group. ( B ) The trends of CD4 + IL-17 + Th17 cells and IL-17 in ATP group. ( C ) The trends of CD4 + CD25 + Foxp3 + Treg cells and TGF-β 1 in ATP group. ( D ) The trends of CD4 + CD25 + Foxp3 + Treg cells and CD4 + IL-17 + Th17 cells in ATP group. ( E ) The ratios of Treg/Th17 among ATP group, BBG group and Control group. * P<0.05, ATP group vs BBG group, # P<0.05, ATP group vs Control group.

    Article Snippet: The treated spleen cells (1×10^6cells) were stained with anti-P2X7 receptor (extracellular)-FITC antibody (Alomone, Jerusalem, Israel) and CD68 antibody (NOVUS, Littleton, USA), and the white cell suspension obtained from the spleen was stained with FITC conjugated mouse anti-rat CD4, PE conjugated mouse anti-rat CD25 (BD Bioscience, MD, USA), PE conjugated anti-IL-17A and APC conjugated anti-Foxp3 antibodies (eBioscience, San Jose, CA, USA).

    Techniques:

    P2X7 receptors identified by immunofluorescence, in renal cortex and medulla in Sham and Ang II-infused rats. The expression of P2X7 receptors in the Ang II group is localized in tubular membranes ( A ). Western blot ( B ) showed abundance of the P2X7 receptor protein in the Ang II group ( n = 7 per group). * p < 0.01 vs. Sham. Ang II = angiotensin II, BBG = Brilliant blue G.

    Journal: International Journal of Molecular Sciences

    Article Title: The Role of P2X7 Purinergic Receptors in the Renal Inflammation Associated with Angiotensin II-Induced Hypertension

    doi: 10.3390/ijms21114041

    Figure Lengend Snippet: P2X7 receptors identified by immunofluorescence, in renal cortex and medulla in Sham and Ang II-infused rats. The expression of P2X7 receptors in the Ang II group is localized in tubular membranes ( A ). Western blot ( B ) showed abundance of the P2X7 receptor protein in the Ang II group ( n = 7 per group). * p < 0.01 vs. Sham. Ang II = angiotensin II, BBG = Brilliant blue G.

    Article Snippet: Kidney sections were incubated with 10% blocking serum in PBS for 1 h at room temperature followed by incubation at 4 °C overnight with the fluorescein isothiocyanate (FITC)-labeled antibodies anti-P2X7-FITC (Cat #: APR-008-F, rabbit polyclonal antibody, Alomone Laboratories, Jerusalem, Israel) at 1:250 [ ].

    Techniques: Immunofluorescence, Expressing, Western Blot

    P2X7 receptors (second column) in the infiltrating T cells of Ang II group co-localize with cluster differentiation (CD, first column) antigens in T cells (CD 3 positive cells), B cells (CD5 and CD20 positive cells), macrophages (CD68 positive cells) and leukocyte common antigen CD45, predominantly expressed in T lymphocytes.

    Journal: International Journal of Molecular Sciences

    Article Title: The Role of P2X7 Purinergic Receptors in the Renal Inflammation Associated with Angiotensin II-Induced Hypertension

    doi: 10.3390/ijms21114041

    Figure Lengend Snippet: P2X7 receptors (second column) in the infiltrating T cells of Ang II group co-localize with cluster differentiation (CD, first column) antigens in T cells (CD 3 positive cells), B cells (CD5 and CD20 positive cells), macrophages (CD68 positive cells) and leukocyte common antigen CD45, predominantly expressed in T lymphocytes.

    Article Snippet: Kidney sections were incubated with 10% blocking serum in PBS for 1 h at room temperature followed by incubation at 4 °C overnight with the fluorescein isothiocyanate (FITC)-labeled antibodies anti-P2X7-FITC (Cat #: APR-008-F, rabbit polyclonal antibody, Alomone Laboratories, Jerusalem, Israel) at 1:250 [ ].

    Techniques:

    P24XR and P2X7R Mediate ATP-Dependent Calcium Signal Propagation (A and B) Murine RAW 264.7 macrophages were loaded with photoactivable caged-IP 3 and the fluorescent calcium indicator Fluo-4 (green), and calcium signal propagation after IP 3 uncaging in the origin cell (white box) was monitored in live imaging. Experiments were performed in HBSS with 2 mM Ca 2+ (A) or in calcium-free HBSS supplemented with 2 mM EGTA (B). Scale bars, 50 μm. See also . (C and D) Quantification of 3 independent live calcium imaging experiments with RAW 264.7 (C) or primary BMDMs (D). Error bars represent SEM. For statistical data analysis, Student’s t test was used ( ∗ p < 0.05). (E and F) Relative mRNA expression of different members of the P2X family of receptors, measured by real-time PCR in the RAW 264.7 cell line (E) and in BMDM primary macrophages (F). Error bars represent SEM. (G) Quantification of 5 independent live calcium imaging experiments with RAW 264.7 cells pre-treated with the P2X4R inhibitor 5BDBD (100 μM, 30 min at 37°C), the P2X7R inhibitor A740003 (100 μM, 30 min at 37°C), or their vehicle (DMSO) or left untreated. Error bars represent SEM. For statistical data analysis, One-way ANOVA followed by Bonferroni’s multiple comparisons test was used ( ∗∗ p < 0.01; ns, non-significant). (H) Representative western blot (left) and quantification of repeated experiments (right) of P2X4R and P2X7R expression in RAW 264.7 cells transfected with siRNA specific for P2X4R and P2X7R or with scramble siRNA. Control cells were electroporated in the absence of oligonucleotides. (I) Quantification of 3 independent live calcium imaging experiments with RAW 264.7 cells silenced for P2X4R and P2X7R. Error bars represent SEM. For statistical data analysis, one-way ANOVA followed by Bonferroni’s multiple comparisons test was used ( ∗∗ p < 0.01; ns, non-significant).

    Journal: Cell Reports

    Article Title: Intercellular Calcium Signaling Induced by ATP Potentiates Macrophage Phagocytosis

    doi: 10.1016/j.celrep.2019.03.011

    Figure Lengend Snippet: P24XR and P2X7R Mediate ATP-Dependent Calcium Signal Propagation (A and B) Murine RAW 264.7 macrophages were loaded with photoactivable caged-IP 3 and the fluorescent calcium indicator Fluo-4 (green), and calcium signal propagation after IP 3 uncaging in the origin cell (white box) was monitored in live imaging. Experiments were performed in HBSS with 2 mM Ca 2+ (A) or in calcium-free HBSS supplemented with 2 mM EGTA (B). Scale bars, 50 μm. See also . (C and D) Quantification of 3 independent live calcium imaging experiments with RAW 264.7 (C) or primary BMDMs (D). Error bars represent SEM. For statistical data analysis, Student’s t test was used ( ∗ p < 0.05). (E and F) Relative mRNA expression of different members of the P2X family of receptors, measured by real-time PCR in the RAW 264.7 cell line (E) and in BMDM primary macrophages (F). Error bars represent SEM. (G) Quantification of 5 independent live calcium imaging experiments with RAW 264.7 cells pre-treated with the P2X4R inhibitor 5BDBD (100 μM, 30 min at 37°C), the P2X7R inhibitor A740003 (100 μM, 30 min at 37°C), or their vehicle (DMSO) or left untreated. Error bars represent SEM. For statistical data analysis, One-way ANOVA followed by Bonferroni’s multiple comparisons test was used ( ∗∗ p < 0.01; ns, non-significant). (H) Representative western blot (left) and quantification of repeated experiments (right) of P2X4R and P2X7R expression in RAW 264.7 cells transfected with siRNA specific for P2X4R and P2X7R or with scramble siRNA. Control cells were electroporated in the absence of oligonucleotides. (I) Quantification of 3 independent live calcium imaging experiments with RAW 264.7 cells silenced for P2X4R and P2X7R. Error bars represent SEM. For statistical data analysis, one-way ANOVA followed by Bonferroni’s multiple comparisons test was used ( ∗∗ p < 0.01; ns, non-significant).

    Article Snippet: After counting, cells were incubated with anti-CD16/CD32 (BD PharMingen), and subsequently stained with the appropriate combinations of the following antibodies: anti-Cd11b-PerCP Cy5.5 (M1/70, BD Biosciences), anti-CD169-AlexaFluor647 (MOMA-1, Biorad), anti-P2X7R-extracellular-FITC (Alomone Labs), purified anti-P2X4R-extracellular (Alomone Labs) followed by incubation with the secondary anti-rabbit-FITC antibody (Thermo Fisher).

    Techniques: Imaging, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Transfection

    Macrophage Polarization Status Affects Calcium Signal Propagation (A) The surface expression of P2X4R (top) and P2X7R (bottom) was analyzed by flow cytometry in resting, IFNγ-treated (10 ng/mL, 24 h), or IL4-treated (20 ng/mL, 24 h) macrophages. Histograms show the quantification 3 independent biological replicates. Error bars represent SEM. For data analysis, one-way ANOVA followed by Bonferroni’s multiple comparisons test was used (ns, non-significant; ∗∗ p < 0.01; ∗∗∗ p < 0.001). (B) Maximal back-projection of two representative live calcium imaging experiments performed with IFNγ- or IL4-treated RAW 264.7 cells loaded with caged-IP 3 and Fluo-4. The fluorescence variation 60 s after the irradiation of the origin cell (white box) is represented in false colors. (C) Representative traces of live calcium imaging experiments, showing the fluorescence variation after the uncaging of the origin cell (red) and the bystander macrophages (black). (D) Quantification of 3 independent live calcium imaging experiments. Error bars represent SEM. For data analysis, one-way ANOVA followed by Bonferroni’s multiple comparisons test was used ( ∗∗ p < 0.01; ns, non-significant).

    Journal: Cell Reports

    Article Title: Intercellular Calcium Signaling Induced by ATP Potentiates Macrophage Phagocytosis

    doi: 10.1016/j.celrep.2019.03.011

    Figure Lengend Snippet: Macrophage Polarization Status Affects Calcium Signal Propagation (A) The surface expression of P2X4R (top) and P2X7R (bottom) was analyzed by flow cytometry in resting, IFNγ-treated (10 ng/mL, 24 h), or IL4-treated (20 ng/mL, 24 h) macrophages. Histograms show the quantification 3 independent biological replicates. Error bars represent SEM. For data analysis, one-way ANOVA followed by Bonferroni’s multiple comparisons test was used (ns, non-significant; ∗∗ p < 0.01; ∗∗∗ p < 0.001). (B) Maximal back-projection of two representative live calcium imaging experiments performed with IFNγ- or IL4-treated RAW 264.7 cells loaded with caged-IP 3 and Fluo-4. The fluorescence variation 60 s after the irradiation of the origin cell (white box) is represented in false colors. (C) Representative traces of live calcium imaging experiments, showing the fluorescence variation after the uncaging of the origin cell (red) and the bystander macrophages (black). (D) Quantification of 3 independent live calcium imaging experiments. Error bars represent SEM. For data analysis, one-way ANOVA followed by Bonferroni’s multiple comparisons test was used ( ∗∗ p < 0.01; ns, non-significant).

    Article Snippet: After counting, cells were incubated with anti-CD16/CD32 (BD PharMingen), and subsequently stained with the appropriate combinations of the following antibodies: anti-Cd11b-PerCP Cy5.5 (M1/70, BD Biosciences), anti-CD169-AlexaFluor647 (MOMA-1, Biorad), anti-P2X7R-extracellular-FITC (Alomone Labs), purified anti-P2X4R-extracellular (Alomone Labs) followed by incubation with the secondary anti-rabbit-FITC antibody (Thermo Fisher).

    Techniques: Expressing, Flow Cytometry, Imaging, Fluorescence, Irradiation

    Extracellular ATP Is Required for Efficient Phagocytosis (A) Primary BMDMs were incubated with PhRodo E. coli fluorescent bioparticles in the presence or absence of 5 mM EGTA to chelate extracellular calcium. Phagocytosis was monitored at 15 or 30 min by flow cytometry (see <xref ref-type=Figure S4 ). Macrophages incubated with 20 μM cytochalasin D were used as negative reference. The phagocytic index was calculated as the percentage of fluorescent macrophages multiplied by their mean of fluorescence (MFI) and normalized on the cytochalasin-treated samples. (B) Primary BMDMs were loaded with the intracellular calcium chelator BAPTA-AM or its vehicle (loading solution) before performing the phagocytosis assay. (C) Primary BMDMs were incubated with PhRodo E. coli , PhRodo Zymosan, or PhRodo S. aureus fluorescent bioparticles, in the presence or absence of apyrase (5 U/mL). (D) Primary BMDMs were pretreated with the P2X4R inhibitor 5BDBD (100 μM), the P2X7R inhibitor A740003 (100 μM), or their vehicle (DMSO), or were left untreated, before performing the phagocytosis assay. (E) Phagocytosis was performed for 30 min in the presence or absence of MSC-derived EVs, pre-incubated or not with ARL-67516 (30 min, 200 μM). The graphs are representative of at least 3 independent biological replicates, each performed in technical triplicate. Error bars represent SEM. For data analysis, a two-way ANOVA followed by Tukey’s multiple comparisons test was used ( ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ns, non-significant). " width="100%" height="100%">

    Journal: Cell Reports

    Article Title: Intercellular Calcium Signaling Induced by ATP Potentiates Macrophage Phagocytosis

    doi: 10.1016/j.celrep.2019.03.011

    Figure Lengend Snippet: Extracellular ATP Is Required for Efficient Phagocytosis (A) Primary BMDMs were incubated with PhRodo E. coli fluorescent bioparticles in the presence or absence of 5 mM EGTA to chelate extracellular calcium. Phagocytosis was monitored at 15 or 30 min by flow cytometry (see Figure S4 ). Macrophages incubated with 20 μM cytochalasin D were used as negative reference. The phagocytic index was calculated as the percentage of fluorescent macrophages multiplied by their mean of fluorescence (MFI) and normalized on the cytochalasin-treated samples. (B) Primary BMDMs were loaded with the intracellular calcium chelator BAPTA-AM or its vehicle (loading solution) before performing the phagocytosis assay. (C) Primary BMDMs were incubated with PhRodo E. coli , PhRodo Zymosan, or PhRodo S. aureus fluorescent bioparticles, in the presence or absence of apyrase (5 U/mL). (D) Primary BMDMs were pretreated with the P2X4R inhibitor 5BDBD (100 μM), the P2X7R inhibitor A740003 (100 μM), or their vehicle (DMSO), or were left untreated, before performing the phagocytosis assay. (E) Phagocytosis was performed for 30 min in the presence or absence of MSC-derived EVs, pre-incubated or not with ARL-67516 (30 min, 200 μM). The graphs are representative of at least 3 independent biological replicates, each performed in technical triplicate. Error bars represent SEM. For data analysis, a two-way ANOVA followed by Tukey’s multiple comparisons test was used ( ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ns, non-significant).

    Article Snippet: After counting, cells were incubated with anti-CD16/CD32 (BD PharMingen), and subsequently stained with the appropriate combinations of the following antibodies: anti-Cd11b-PerCP Cy5.5 (M1/70, BD Biosciences), anti-CD169-AlexaFluor647 (MOMA-1, Biorad), anti-P2X7R-extracellular-FITC (Alomone Labs), purified anti-P2X4R-extracellular (Alomone Labs) followed by incubation with the secondary anti-rabbit-FITC antibody (Thermo Fisher).

    Techniques: Incubation, Flow Cytometry, Fluorescence, Phagocytosis Assay, Derivative Assay

    Journal: Cell Reports

    Article Title: Intercellular Calcium Signaling Induced by ATP Potentiates Macrophage Phagocytosis

    doi: 10.1016/j.celrep.2019.03.011

    Figure Lengend Snippet:

    Article Snippet: After counting, cells were incubated with anti-CD16/CD32 (BD PharMingen), and subsequently stained with the appropriate combinations of the following antibodies: anti-Cd11b-PerCP Cy5.5 (M1/70, BD Biosciences), anti-CD169-AlexaFluor647 (MOMA-1, Biorad), anti-P2X7R-extracellular-FITC (Alomone Labs), purified anti-P2X4R-extracellular (Alomone Labs) followed by incubation with the secondary anti-rabbit-FITC antibody (Thermo Fisher).

    Techniques: Recombinant, Negative Control, Real-time Polymerase Chain Reaction, Software

    Journal: Cell Reports

    Article Title: Intercellular Calcium Signaling Induced by ATP Potentiates Macrophage Phagocytosis

    doi: 10.1016/j.celrep.2019.03.011

    Figure Lengend Snippet:

    Article Snippet: After counting, cells were incubated with anti-CD16/CD32 (BD PharMingen), and subsequently stained with the appropriate combinations of the following antibodies: anti-Cd11b-PerCP Cy5.5 (M1/70, BD Biosciences), anti-CD169-AlexaFluor647 (MOMA-1, Biorad), anti-P2X7R-extracellular-FITC (Alomone Labs), purified anti-P2X4R-extracellular (Alomone Labs) followed by incubation with the secondary anti-rabbit-FITC antibody (Thermo Fisher).

    Techniques:

    Model of pore former induced lysis. A bacterial toxin inserts a large channel or pore into the erythrocyte membrane. ATP is immediately released through the pore and activates P2X receptors. The membrane insertion of the toxin also causes a steep rise in the intracellular Ca 2+ concentration ([Ca 2+ ] i ), which results from Ca 2+ passing through the pore itself and from activation of P2X receptors, which are non-selective cation channels permeable to Ca 2+ . The increase in [Ca 2+ ] i activates the Ca 2+ -sensitive K + channel K Ca 3.1 and Cl − channel TMEM16A, which results in K + and Cl − efflux and cell shrinkage as obligated water follows. The cells will remain shrunken as long as the K + efflux surpasses the Na + influx via the toxin pore and the P2X receptors. Prolonged stimulation of P2X 7 can activate pannexins, which will contribute to the Na + influx. Eventually, the Na + influx will exceed the K + efflux and the cells will swell and eventually burst. Blockage of the P2X 1 and P2X 7 receptor has been proven as a protective measure for bacterial toxins, and for complement to carry out their toxicity. The model based on previous work [ , , , ]

    Journal: Critical Care

    Article Title: Erythrocyte P2X 1 receptor expression is correlated with change in haematocrit in patients admitted to the ICU with blood pathogen-positive sepsis

    doi: 10.1186/s13054-018-2100-3

    Figure Lengend Snippet: Model of pore former induced lysis. A bacterial toxin inserts a large channel or pore into the erythrocyte membrane. ATP is immediately released through the pore and activates P2X receptors. The membrane insertion of the toxin also causes a steep rise in the intracellular Ca 2+ concentration ([Ca 2+ ] i ), which results from Ca 2+ passing through the pore itself and from activation of P2X receptors, which are non-selective cation channels permeable to Ca 2+ . The increase in [Ca 2+ ] i activates the Ca 2+ -sensitive K + channel K Ca 3.1 and Cl − channel TMEM16A, which results in K + and Cl − efflux and cell shrinkage as obligated water follows. The cells will remain shrunken as long as the K + efflux surpasses the Na + influx via the toxin pore and the P2X receptors. Prolonged stimulation of P2X 7 can activate pannexins, which will contribute to the Na + influx. Eventually, the Na + influx will exceed the K + efflux and the cells will swell and eventually burst. Blockage of the P2X 1 and P2X 7 receptor has been proven as a protective measure for bacterial toxins, and for complement to carry out their toxicity. The model based on previous work [ , , , ]

    Article Snippet: Cells in each sample were counted on a cell counter (Spectre, Merch Millipore, USA) prior to exposure to either P2X 1 (catalogue number APR-022-AG) or P2X 7 (catalogue number APR-008-F) fluorescein isothiocyanate (FITC)-conjugated antibody (Alomone Labs, Jerusalem, Israel) for 1 h at room temperature in the dark at 200 rpm in concentrations according to the manufacturer’s instructions (10 mg antibody to 10 6 cells).

    Techniques: Lysis, Concentration Assay, Activation Assay

    Freeze-thaw - control experiments. a Whole blood drawn from healthy volunteers was incubated either with vehicle (Veh), 100 μM NF449 (P2X 1 antagonist) or 100 μM A804598 (P2X 7 antagonist) and frozen and stored for 3 weeks as whole blood at − 80 °C. After thawing, lysis was measured as absorbance of free haemoglobin in supernatant at 540 nm. n = 3, mean ± SEM. b Flow cytometry of thawed whole blood samples diluted 1000-fold, to ascertain equal distribution of blood cell sub-populations. Cells were identified by forward scatter FSC and side scatter SSC and quantified based on fixed regions. c Bar graph shows data presented as mean ± SEM, n = 3. ns, not significant

    Journal: Critical Care

    Article Title: Erythrocyte P2X 1 receptor expression is correlated with change in haematocrit in patients admitted to the ICU with blood pathogen-positive sepsis

    doi: 10.1186/s13054-018-2100-3

    Figure Lengend Snippet: Freeze-thaw - control experiments. a Whole blood drawn from healthy volunteers was incubated either with vehicle (Veh), 100 μM NF449 (P2X 1 antagonist) or 100 μM A804598 (P2X 7 antagonist) and frozen and stored for 3 weeks as whole blood at − 80 °C. After thawing, lysis was measured as absorbance of free haemoglobin in supernatant at 540 nm. n = 3, mean ± SEM. b Flow cytometry of thawed whole blood samples diluted 1000-fold, to ascertain equal distribution of blood cell sub-populations. Cells were identified by forward scatter FSC and side scatter SSC and quantified based on fixed regions. c Bar graph shows data presented as mean ± SEM, n = 3. ns, not significant

    Article Snippet: Cells in each sample were counted on a cell counter (Spectre, Merch Millipore, USA) prior to exposure to either P2X 1 (catalogue number APR-022-AG) or P2X 7 (catalogue number APR-008-F) fluorescein isothiocyanate (FITC)-conjugated antibody (Alomone Labs, Jerusalem, Israel) for 1 h at room temperature in the dark at 200 rpm in concentrations according to the manufacturer’s instructions (10 mg antibody to 10 6 cells).

    Techniques: Incubation, Lysis, Flow Cytometry

    Correlation statistics

    Journal: Critical Care

    Article Title: Erythrocyte P2X 1 receptor expression is correlated with change in haematocrit in patients admitted to the ICU with blood pathogen-positive sepsis

    doi: 10.1186/s13054-018-2100-3

    Figure Lengend Snippet: Correlation statistics

    Article Snippet: Cells in each sample were counted on a cell counter (Spectre, Merch Millipore, USA) prior to exposure to either P2X 1 (catalogue number APR-022-AG) or P2X 7 (catalogue number APR-008-F) fluorescein isothiocyanate (FITC)-conjugated antibody (Alomone Labs, Jerusalem, Israel) for 1 h at room temperature in the dark at 200 rpm in concentrations according to the manufacturer’s instructions (10 mg antibody to 10 6 cells).

    Techniques: Activity Assay, Expressing

    Haematocrit and haemoglobin levels and erythrocyte P2X 7 receptor expression. a Change in haematocrit in blood pathogen-positive patients with sepsis between Emergency Department (ER) and ICU admission and P2X 7 receptor expression on erythrocytes were not correlated. The right panel shows the change in haematocrit in blood pathogen-positive patients grouped by high or low expression of P2X 7 (Δhct/hour). b Change in haematocrit in blood pathogen-negative patients with sepsis between ER and ICU admission and P2X 7 receptor expression on erythrocytes. Right panel shows the change in haematocrit in blood pathogen-negative patients grouped by high or low expression of P2X 7 (Δhct/hour). c Change in haemoglobin in blood pathogen-positive patients with sepsis between ER and ICU admission and P2X 7 receptor expression on erythrocytes were not correlated. The right panel shows change in haemoglobin in blood pathogen-positive patients grouped by high or low expression of P2X 7 (ΔHgb/hour, not significant (ns)). d Change in haemoglobin in blood pathogen-negative patients with sepsis between ER and ICU admission and P2X 7 receptor expression on erythrocytes. The right panel shows change in haemoglobin in blood pathogen-negative patients grouped by high or low expression of P2X 7 (ΔHgb/hour)

    Journal: Critical Care

    Article Title: Erythrocyte P2X 1 receptor expression is correlated with change in haematocrit in patients admitted to the ICU with blood pathogen-positive sepsis

    doi: 10.1186/s13054-018-2100-3

    Figure Lengend Snippet: Haematocrit and haemoglobin levels and erythrocyte P2X 7 receptor expression. a Change in haematocrit in blood pathogen-positive patients with sepsis between Emergency Department (ER) and ICU admission and P2X 7 receptor expression on erythrocytes were not correlated. The right panel shows the change in haematocrit in blood pathogen-positive patients grouped by high or low expression of P2X 7 (Δhct/hour). b Change in haematocrit in blood pathogen-negative patients with sepsis between ER and ICU admission and P2X 7 receptor expression on erythrocytes. Right panel shows the change in haematocrit in blood pathogen-negative patients grouped by high or low expression of P2X 7 (Δhct/hour). c Change in haemoglobin in blood pathogen-positive patients with sepsis between ER and ICU admission and P2X 7 receptor expression on erythrocytes were not correlated. The right panel shows change in haemoglobin in blood pathogen-positive patients grouped by high or low expression of P2X 7 (ΔHgb/hour, not significant (ns)). d Change in haemoglobin in blood pathogen-negative patients with sepsis between ER and ICU admission and P2X 7 receptor expression on erythrocytes. The right panel shows change in haemoglobin in blood pathogen-negative patients grouped by high or low expression of P2X 7 (ΔHgb/hour)

    Article Snippet: Cells in each sample were counted on a cell counter (Spectre, Merch Millipore, USA) prior to exposure to either P2X 1 (catalogue number APR-022-AG) or P2X 7 (catalogue number APR-008-F) fluorescein isothiocyanate (FITC)-conjugated antibody (Alomone Labs, Jerusalem, Israel) for 1 h at room temperature in the dark at 200 rpm in concentrations according to the manufacturer’s instructions (10 mg antibody to 10 6 cells).

    Techniques: Expressing

    (A) P2X7R and NLRP3 immunoprecipitation (IP) in human CD4+ T cells. Expression of NLRP3 (top blot) and P2X7R (bottom blot) is shown. Lane 1: Total protein. Lane 2: IP with NLRP3 Ab. Lane 3: IP with P2X7R Ab. Lane 4: IP with Ab alone (NLRP3 and P2X7R). Lane 5: IP with control IgG (for NLRP3 Ab in top blot, for P2X7R Ab in bottom blot). The experiment was run in triplicate (representative blot shown). (B and C) Confocal microscopy analysis (B, scale bar: 5 μm, ×100 original magnification; C, scale bars: 20 μm, ×40 original magnification) depicting baseline colocalization of P2X7R (green) and NLRP3 (red) in human CD4+ T cells. Cells were stained with DAPI (blue) and immunolabeled with anti-P2X7R (green) and anti-NLRP3 Abs (red) (n = 3). (D–F) Bar graphs depicting expression of NLRP3 mRNA by qRT-PCR (D), and protein by flow cytometry (E) and ELISA (F), evaluated in human CD4+ T cells activated with benzoyl ATP (BzATP) and treated with CE-224,535, a P2X7R inhibitor. Experiments were run in duplicate (n = 5). (G) Bar graph representing expression of NLRP3 on human CD4+P2X7R+ cells analyzed by flow cytometry upon BzATP stimulation (n = 5). (H) Representative flow dot plots of NLRP3 expression upon gating on human BzATP-stimulated CD4+P2X7R+ cells. (I) Confocal analysis (scale bar: 5 μm; ×100 original magnification) depicting colocalization of P2X7R (green) and NLRP3 (red) in CD4+ T cells upon in vitro stimulation of P2X7R with BzATP (n = 3). (J–M) Bar graphs comparing expression of NLRP3 downstream signaling Th2-related factors IL-4 (J), IRF4 (K), GATA-3 (L), and IL-10 (M) by qRT-PCR using mRNA isolated from human CD4+ T cells activated with BzATP and treated with the P2X7R inhibitor CE-224,535. Experiments were run in triplicate (n = 5). Data are expressed as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; 1-way ANOVA with Bonferroni’s post hoc test or Student’s t test. mRNA expression was normalized to β-actin (ACTB).

    Journal: The Journal of Clinical Investigation

    Article Title: P2X7R mutation disrupts the NLRP3-mediated Th program and predicts poor cardiac allograft outcomes

    doi: 10.1172/JCI94524

    Figure Lengend Snippet: (A) P2X7R and NLRP3 immunoprecipitation (IP) in human CD4+ T cells. Expression of NLRP3 (top blot) and P2X7R (bottom blot) is shown. Lane 1: Total protein. Lane 2: IP with NLRP3 Ab. Lane 3: IP with P2X7R Ab. Lane 4: IP with Ab alone (NLRP3 and P2X7R). Lane 5: IP with control IgG (for NLRP3 Ab in top blot, for P2X7R Ab in bottom blot). The experiment was run in triplicate (representative blot shown). (B and C) Confocal microscopy analysis (B, scale bar: 5 μm, ×100 original magnification; C, scale bars: 20 μm, ×40 original magnification) depicting baseline colocalization of P2X7R (green) and NLRP3 (red) in human CD4+ T cells. Cells were stained with DAPI (blue) and immunolabeled with anti-P2X7R (green) and anti-NLRP3 Abs (red) (n = 3). (D–F) Bar graphs depicting expression of NLRP3 mRNA by qRT-PCR (D), and protein by flow cytometry (E) and ELISA (F), evaluated in human CD4+ T cells activated with benzoyl ATP (BzATP) and treated with CE-224,535, a P2X7R inhibitor. Experiments were run in duplicate (n = 5). (G) Bar graph representing expression of NLRP3 on human CD4+P2X7R+ cells analyzed by flow cytometry upon BzATP stimulation (n = 5). (H) Representative flow dot plots of NLRP3 expression upon gating on human BzATP-stimulated CD4+P2X7R+ cells. (I) Confocal analysis (scale bar: 5 μm; ×100 original magnification) depicting colocalization of P2X7R (green) and NLRP3 (red) in CD4+ T cells upon in vitro stimulation of P2X7R with BzATP (n = 3). (J–M) Bar graphs comparing expression of NLRP3 downstream signaling Th2-related factors IL-4 (J), IRF4 (K), GATA-3 (L), and IL-10 (M) by qRT-PCR using mRNA isolated from human CD4+ T cells activated with BzATP and treated with the P2X7R inhibitor CE-224,535. Experiments were run in triplicate (n = 5). Data are expressed as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; 1-way ANOVA with Bonferroni’s post hoc test or Student’s t test. mRNA expression was normalized to β-actin (ACTB).

    Article Snippet: FITC-conjugated anti–mouse P2X7R (APR-008-F) was purchased from Alomone Labs. Alexa Fluor 700–conjugated anti–human NLRP3 (IC7578N) was purchased from R&D Systems.

    Techniques: Immunoprecipitation, Expressing, Confocal Microscopy, Staining, Immunolabeling, Quantitative RT-PCR, Flow Cytometry, Enzyme-linked Immunosorbent Assay, In Vitro, Isolation

    (A) A 3D representation of the full-length structure of P2X7R, highlighting the putative location of the P2X7R mutation in the C-terminal intracellular portion. (B and C) Quantification of P2X7R total protein (B, ELISA, n = 3) and of P2X7R mRNA (C, qRT-PCR, n = 10) on CD4+ T cells of carrier and noncarrier patients. Samples were run in duplicate (B) or in triplicate (C) and normalized to expression level of β-actin (ACTB). (D) Transcriptome profiling of immune-relevant genes (see also Supplemental Table 3) examined in CD4+ T cells of carrier and noncarrier cardiac-transplanted patients (n = 5). (E–G) Expression of NLRP3 mRNA using qRT-PCR (E) and NLRP3 protein using flow cytometry (F) and ELISA (G) in CD4+ T cells of carrier and noncarrier patients (n = 5). (H and I) Flow cytometric expression of NLRP3 on CD4+P2X7R+ cells of carrier patients stimulated with BzATP (n = 5). (J) Percentage of P2X7R+NLRP3+ cells of carrier and noncarrier patients analyzed by immunofluorescence (Figure 1C and Supplemental Figure 2G) (n = 3). (K) Confocal microscopy analysis (×100 original magnification) of P2X7R (green) and NLRP3 (red) coexpression in CD4+ T cells of carrier patients (n = 3). Scale bar: 5 μm. (L) Subcellular localization of NLRP3 in CD4+ T cells of carrier and of noncarrier patients (n = 3). (M and N) IL-4 (M) and IRF4 (N) gene expression detected after ChIP with NLRP3 antibody in CD4+ T cells. (n = 3). (O) Quantification of NLRP3 protein measured in CD4+ T cells treated with the ubiquitin/protease inhibitor MG132 (n = 3). Bars represent mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; Student’s t test or 2-way ANOVA with Bonferroni’s post hoc test.

    Journal: The Journal of Clinical Investigation

    Article Title: P2X7R mutation disrupts the NLRP3-mediated Th program and predicts poor cardiac allograft outcomes

    doi: 10.1172/JCI94524

    Figure Lengend Snippet: (A) A 3D representation of the full-length structure of P2X7R, highlighting the putative location of the P2X7R mutation in the C-terminal intracellular portion. (B and C) Quantification of P2X7R total protein (B, ELISA, n = 3) and of P2X7R mRNA (C, qRT-PCR, n = 10) on CD4+ T cells of carrier and noncarrier patients. Samples were run in duplicate (B) or in triplicate (C) and normalized to expression level of β-actin (ACTB). (D) Transcriptome profiling of immune-relevant genes (see also Supplemental Table 3) examined in CD4+ T cells of carrier and noncarrier cardiac-transplanted patients (n = 5). (E–G) Expression of NLRP3 mRNA using qRT-PCR (E) and NLRP3 protein using flow cytometry (F) and ELISA (G) in CD4+ T cells of carrier and noncarrier patients (n = 5). (H and I) Flow cytometric expression of NLRP3 on CD4+P2X7R+ cells of carrier patients stimulated with BzATP (n = 5). (J) Percentage of P2X7R+NLRP3+ cells of carrier and noncarrier patients analyzed by immunofluorescence (Figure 1C and Supplemental Figure 2G) (n = 3). (K) Confocal microscopy analysis (×100 original magnification) of P2X7R (green) and NLRP3 (red) coexpression in CD4+ T cells of carrier patients (n = 3). Scale bar: 5 μm. (L) Subcellular localization of NLRP3 in CD4+ T cells of carrier and of noncarrier patients (n = 3). (M and N) IL-4 (M) and IRF4 (N) gene expression detected after ChIP with NLRP3 antibody in CD4+ T cells. (n = 3). (O) Quantification of NLRP3 protein measured in CD4+ T cells treated with the ubiquitin/protease inhibitor MG132 (n = 3). Bars represent mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; Student’s t test or 2-way ANOVA with Bonferroni’s post hoc test.

    Article Snippet: FITC-conjugated anti–mouse P2X7R (APR-008-F) was purchased from Alomone Labs. Alexa Fluor 700–conjugated anti–human NLRP3 (IC7578N) was purchased from R&D Systems.

    Techniques: Mutagenesis, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Expressing, Flow Cytometry, Immunofluorescence, Confocal Microscopy, Protease Inhibitor

    (A) P2X7R–/– mice receiving bm12 heart transplantation demonstrated reduced graft survival as compared with B6 recipients (**P < 0.01), which was significantly prolonged by anti–IL-17 treatment (murine IL-17–depleting antibody) (*P < 0.05 vs. P2X7R–/–) (n = 10 mice per group). (B–D) Semiquantification of graft infiltration (B), coronary vasculopathy (C), and myocyte necrosis (D) confirmed accelerated allograft rejection in P2X7R–/– mice (n = 3). (E) Representative H&E staining (x20 original magnification) showing graft cell infiltration (top panels), vasculopathy (middle panels), and myocyte necrosis (bottom panels) in B6 and P2X7R–/– mice. Scale bars: 200 μm (middle panels), 300 μm (top and bottom panels). (F and G) Numbers of IFN-γ–producing (F) and IL-4–producing (G) cells (ELISPOT) measured in cardiac-transplanted mice (n = 3). (H–M) Percentage of CD4+IL-17+ (H), CD4+IFN-γ+ (I), CD4+IL-10+ (J), CD4+CD44hiCD62Llo (K), CD8+CD44hiCD62Llo (L), and CD4+CD25+Foxp3+ (M) cells detected by flow cytometry in B6 and P2X7R–/– cardiac-transplanted mice and in P2X7R–/– anti–IL-17–treated mice (n = 5). (N) Serum IL-17 level (Luminex) measured in B6 and P2X7R–/– cardiac-transplanted mice and in P2X7R–/– anti–IL-17–treated mice (n = 5). (O) Percentage of CD4+NLRP3+ cells analyzed by flow cytometry in P2X7R–/– and B6 mice (n = 3). (P) Number of IL-4–producing cells (ELISPOT) in P2X7R–/– and B6 mice upon allostimulation (n = 3). (Q) Serum IL-4 level (Luminex), measured in B6 and P2X7R–/– cardiac-transplanted mice (n = 5). Samples were run in duplicate (Luminex) and in triplicate (ELISPOT). Bars represent mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.001; log-rank (Mantel-Cox) test (A), Wilcoxon’s and Student’s t test (2 groups), 1-way ANOVA with Bonferroni’s post hoc test (3 groups).

    Journal: The Journal of Clinical Investigation

    Article Title: P2X7R mutation disrupts the NLRP3-mediated Th program and predicts poor cardiac allograft outcomes

    doi: 10.1172/JCI94524

    Figure Lengend Snippet: (A) P2X7R–/– mice receiving bm12 heart transplantation demonstrated reduced graft survival as compared with B6 recipients (**P < 0.01), which was significantly prolonged by anti–IL-17 treatment (murine IL-17–depleting antibody) (*P < 0.05 vs. P2X7R–/–) (n = 10 mice per group). (B–D) Semiquantification of graft infiltration (B), coronary vasculopathy (C), and myocyte necrosis (D) confirmed accelerated allograft rejection in P2X7R–/– mice (n = 3). (E) Representative H&E staining (x20 original magnification) showing graft cell infiltration (top panels), vasculopathy (middle panels), and myocyte necrosis (bottom panels) in B6 and P2X7R–/– mice. Scale bars: 200 μm (middle panels), 300 μm (top and bottom panels). (F and G) Numbers of IFN-γ–producing (F) and IL-4–producing (G) cells (ELISPOT) measured in cardiac-transplanted mice (n = 3). (H–M) Percentage of CD4+IL-17+ (H), CD4+IFN-γ+ (I), CD4+IL-10+ (J), CD4+CD44hiCD62Llo (K), CD8+CD44hiCD62Llo (L), and CD4+CD25+Foxp3+ (M) cells detected by flow cytometry in B6 and P2X7R–/– cardiac-transplanted mice and in P2X7R–/– anti–IL-17–treated mice (n = 5). (N) Serum IL-17 level (Luminex) measured in B6 and P2X7R–/– cardiac-transplanted mice and in P2X7R–/– anti–IL-17–treated mice (n = 5). (O) Percentage of CD4+NLRP3+ cells analyzed by flow cytometry in P2X7R–/– and B6 mice (n = 3). (P) Number of IL-4–producing cells (ELISPOT) in P2X7R–/– and B6 mice upon allostimulation (n = 3). (Q) Serum IL-4 level (Luminex), measured in B6 and P2X7R–/– cardiac-transplanted mice (n = 5). Samples were run in duplicate (Luminex) and in triplicate (ELISPOT). Bars represent mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.001; log-rank (Mantel-Cox) test (A), Wilcoxon’s and Student’s t test (2 groups), 1-way ANOVA with Bonferroni’s post hoc test (3 groups).

    Article Snippet: FITC-conjugated anti–mouse P2X7R (APR-008-F) was purchased from Alomone Labs. Alexa Fluor 700–conjugated anti–human NLRP3 (IC7578N) was purchased from R&D Systems.

    Techniques: Transplantation Assay, Staining, Enzyme-linked Immunospot, Flow Cytometry, Luminex

    (A) Bar graph depicting the percentage of cardiac-transplanted patients who carry the WT or mutant P2X7R allele (n = 102) with an MIT change greater than 0.5 mm (early cardiac allograft vasculopathy, in black) at 1 year after transplantation in the CTOT-05 cohort. (B) Bar graph depicting the number of acute rejection episodes in cardiac-transplanted patients who carry the WT (black) or mutant (white) P2X7R allele (n = 181) within the first year after transplant in the NIT-Bergamo cohort. (C) Bar graph depicting the percentage of cardiac-transplanted patients who carry the WT or mutant P2X7R allele (n = 130) with major adverse cardiac events (MACEs, in black) at 10 years of follow-up in the AIRT-Bologna cohort. In A and C: black, percentage of patients who experienced the event; white, percentage who were free from events. (D) Line graph depicting the estimated odds ratio (OR) for clinical outcomes recorded in the 3 cohorts of cardiac-transplanted patients who carry the WT or mutant P2X7R allele. In the NIT-Bergamo cohort, the OR was calculated based on the requirement of medical intervention for acute rejection episodes with a frequency of greater or less than 3 episodes. *P < 0.05; **P < 0.01. Supplemental Tables 7–9 report detailed analyses. Fisher’s exact and Student’s t tests. (E and F) A stable connection between P2X7R and NLRP3 is necessary to establish a physiological NLRP3-mediated Th2 program (E), while alteration in the P2X7R intracellular domain induces NLRP3 displacement and retains NLRP3 in the cell membrane, thus preventing its nuclear activity and accelerating ubiquitination of NLRP3 (F). This shifts the balance of the immune response toward Th17 cells and favors the development of immune-related events, such as allograft rejection and vasculopathy. Ub, ubiquitin; eATP, extracellular ATP.

    Journal: The Journal of Clinical Investigation

    Article Title: P2X7R mutation disrupts the NLRP3-mediated Th program and predicts poor cardiac allograft outcomes

    doi: 10.1172/JCI94524

    Figure Lengend Snippet: (A) Bar graph depicting the percentage of cardiac-transplanted patients who carry the WT or mutant P2X7R allele (n = 102) with an MIT change greater than 0.5 mm (early cardiac allograft vasculopathy, in black) at 1 year after transplantation in the CTOT-05 cohort. (B) Bar graph depicting the number of acute rejection episodes in cardiac-transplanted patients who carry the WT (black) or mutant (white) P2X7R allele (n = 181) within the first year after transplant in the NIT-Bergamo cohort. (C) Bar graph depicting the percentage of cardiac-transplanted patients who carry the WT or mutant P2X7R allele (n = 130) with major adverse cardiac events (MACEs, in black) at 10 years of follow-up in the AIRT-Bologna cohort. In A and C: black, percentage of patients who experienced the event; white, percentage who were free from events. (D) Line graph depicting the estimated odds ratio (OR) for clinical outcomes recorded in the 3 cohorts of cardiac-transplanted patients who carry the WT or mutant P2X7R allele. In the NIT-Bergamo cohort, the OR was calculated based on the requirement of medical intervention for acute rejection episodes with a frequency of greater or less than 3 episodes. *P < 0.05; **P < 0.01. Supplemental Tables 7–9 report detailed analyses. Fisher’s exact and Student’s t tests. (E and F) A stable connection between P2X7R and NLRP3 is necessary to establish a physiological NLRP3-mediated Th2 program (E), while alteration in the P2X7R intracellular domain induces NLRP3 displacement and retains NLRP3 in the cell membrane, thus preventing its nuclear activity and accelerating ubiquitination of NLRP3 (F). This shifts the balance of the immune response toward Th17 cells and favors the development of immune-related events, such as allograft rejection and vasculopathy. Ub, ubiquitin; eATP, extracellular ATP.

    Article Snippet: FITC-conjugated anti–mouse P2X7R (APR-008-F) was purchased from Alomone Labs. Alexa Fluor 700–conjugated anti–human NLRP3 (IC7578N) was purchased from R&D Systems.

    Techniques: Mutagenesis, Transplantation Assay, Activity Assay