anti p2x1  (Alomone Labs)


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    Alomone Labs anti p2x1
    Anti P2x1, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti p2x1/product/Alomone Labs
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti p2x1 - by Bioz Stars, 2023-02
    93/100 stars

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    anti p2x1  (Alomone Labs)


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    Alomone Labs anti p2x1
    Anti P2x1, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti p2x1/product/Alomone Labs
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti p2x1 - by Bioz Stars, 2023-02
    93/100 stars

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    anti p2rx1  (Alomone Labs)


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    Alomone Labs anti p2rx1
    a Volcano plots of differential gene expression in 145 primary PDAC, 46 adjacent pancreases, 25 liver metastases and 27 adjacent livers. Red dots represent upregulated immune-related genes, and blue dots represent downregulated immune-related genes. b Immunome analyses of 26 infiltrating immune cell types in adjacent pancreas, primary PDAC, adjacent liver tissue and metastatic PDAC samples. c GO Biological Process analyses of differentially expressed genes between adjacent liver tissue and metastatic PDAC samples. d Expression analyses of <t>P2RX1</t> in the adjacent pancreas, primary PDAC, adjacent liver tissue and metastatic PDAC samples from the GSE71729 and Renji cohorts. e Correlation analyses between P2RX1 and immune checkpoint molecules in metastatic PDAC samples. Bars represent mean ± standard deviation in ( d ). * P < 0.05, ** P < 0.01, and *** P < 0.001, by one-way ANOVA and Tukey’s multiple comparisons test ( d left), or Student’s t test ( d right). Source data are provided as a Source data file.
    Anti P2rx1, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti p2rx1/product/Alomone Labs
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti p2rx1 - by Bioz Stars, 2023-02
    93/100 stars

    Images

    1) Product Images from "Identification of a subset of immunosuppressive P2RX1-negative neutrophils in pancreatic cancer liver metastasis"

    Article Title: Identification of a subset of immunosuppressive P2RX1-negative neutrophils in pancreatic cancer liver metastasis

    Journal: Nature Communications

    doi: 10.1038/s41467-020-20447-y

    a Volcano plots of differential gene expression in 145 primary PDAC, 46 adjacent pancreases, 25 liver metastases and 27 adjacent livers. Red dots represent upregulated immune-related genes, and blue dots represent downregulated immune-related genes. b Immunome analyses of 26 infiltrating immune cell types in adjacent pancreas, primary PDAC, adjacent liver tissue and metastatic PDAC samples. c GO Biological Process analyses of differentially expressed genes between adjacent liver tissue and metastatic PDAC samples. d Expression analyses of P2RX1 in the adjacent pancreas, primary PDAC, adjacent liver tissue and metastatic PDAC samples from the GSE71729 and Renji cohorts. e Correlation analyses between P2RX1 and immune checkpoint molecules in metastatic PDAC samples. Bars represent mean ± standard deviation in ( d ). * P < 0.05, ** P < 0.01, and *** P < 0.001, by one-way ANOVA and Tukey’s multiple comparisons test ( d left), or Student’s t test ( d right). Source data are provided as a Source data file.
    Figure Legend Snippet: a Volcano plots of differential gene expression in 145 primary PDAC, 46 adjacent pancreases, 25 liver metastases and 27 adjacent livers. Red dots represent upregulated immune-related genes, and blue dots represent downregulated immune-related genes. b Immunome analyses of 26 infiltrating immune cell types in adjacent pancreas, primary PDAC, adjacent liver tissue and metastatic PDAC samples. c GO Biological Process analyses of differentially expressed genes between adjacent liver tissue and metastatic PDAC samples. d Expression analyses of P2RX1 in the adjacent pancreas, primary PDAC, adjacent liver tissue and metastatic PDAC samples from the GSE71729 and Renji cohorts. e Correlation analyses between P2RX1 and immune checkpoint molecules in metastatic PDAC samples. Bars represent mean ± standard deviation in ( d ). * P < 0.05, ** P < 0.01, and *** P < 0.001, by one-way ANOVA and Tukey’s multiple comparisons test ( d left), or Student’s t test ( d right). Source data are provided as a Source data file.

    Techniques Used: Expressing, Standard Deviation

    a , b P2rx1 −/− mice were generated using CRISPR/Cas9 system. Schematic diagram was shown in ( a ) and genotyping results were shown in ( b ) (representative result from three independent experiments). c , d KPC cells were intrasplenically injected to seed livers of WT and P2rx1 −/− mice, and in vivo imaging was performed at sequential times. Representative images are shown in ( c ), and quantitative results are shown in ( d ) ( n = 6 per group, three independent experiments). e Representative images of liver metastatic samples harvested at day 17. f Liver weight of liver metastatic samples was measured at day 17 ( n = 5 per group, two independent experiments). g Survival analysis of liver metastatic WT or P2rx1 −/− mice within a duration of 5 weeks ( n = 10 per group, two independent experiments). h Normal liver (D0) and two sequential stages (D3 and D17) of liver metastases in WT and P2rx1 −/− mice were harvested for RNA-seq, and PCA analyses were performed ( n = 3 for D0, n = 4 for D3 and D17). i Representative Ki67 immunohistochemical staining of WT and P2rx1 −/− liver metastases at day 17 ( n = 4 per group, two independent experiments). 100 μm of scale bar for low power fields, 25 μm of scale bar for high power fields. j Heatmap of immune checkpoint molecules in liver metastases of WT or P2rx1 −/− mice at D3 and D17 ( n = 4 per group). Bars represent mean ± standard deviation in ( d , f ). P values are derived from two-sided Student’s t test ( d , f ), or log-rank test ( g ). Source data are provided as a Source data file.
    Figure Legend Snippet: a , b P2rx1 −/− mice were generated using CRISPR/Cas9 system. Schematic diagram was shown in ( a ) and genotyping results were shown in ( b ) (representative result from three independent experiments). c , d KPC cells were intrasplenically injected to seed livers of WT and P2rx1 −/− mice, and in vivo imaging was performed at sequential times. Representative images are shown in ( c ), and quantitative results are shown in ( d ) ( n = 6 per group, three independent experiments). e Representative images of liver metastatic samples harvested at day 17. f Liver weight of liver metastatic samples was measured at day 17 ( n = 5 per group, two independent experiments). g Survival analysis of liver metastatic WT or P2rx1 −/− mice within a duration of 5 weeks ( n = 10 per group, two independent experiments). h Normal liver (D0) and two sequential stages (D3 and D17) of liver metastases in WT and P2rx1 −/− mice were harvested for RNA-seq, and PCA analyses were performed ( n = 3 for D0, n = 4 for D3 and D17). i Representative Ki67 immunohistochemical staining of WT and P2rx1 −/− liver metastases at day 17 ( n = 4 per group, two independent experiments). 100 μm of scale bar for low power fields, 25 μm of scale bar for high power fields. j Heatmap of immune checkpoint molecules in liver metastases of WT or P2rx1 −/− mice at D3 and D17 ( n = 4 per group). Bars represent mean ± standard deviation in ( d , f ). P values are derived from two-sided Student’s t test ( d , f ), or log-rank test ( g ). Source data are provided as a Source data file.

    Techniques Used: Generated, CRISPR, Injection, In Vivo Imaging, RNA Sequencing Assay, Immunohistochemical staining, Staining, Standard Deviation, Derivative Assay

    a Single cell suspension was obtained from mouse spleen and expression of P2RX1 was determined in indicated immune cell types ( n = 3 per group, two independent experiments). Left dotted line indicates the mean of negative control (NC) (secondary antibody only), and right dotted line indicates the mean of Ly6G+ cells. b KPC cells were intrasplenically injected to seed livers of WT mice. A single cell suspension was obtained from liver metastases and adjacent liver tissues of WT mice at day 17. The frequency of CD45+P2RX1+ cells was determined by flow cytometry ( n = 4 per group, three independent experiments). c KPC cells were intrasplenically injected to seed livers of WT mice. Immune cells were enriched from single cell suspension of liver metastases and adjacent liver tissues at day 17. P2RX1 expression in the indicated immune cell types was determined by flow cytometry ( n = 4 per group, three independent experiments). d Representative images of H&E staining and immunofluorescence staining of P2RX1 (Green), Ly6G (Red) and DAPI (Blue) in KPC mice spontaneous liver metastases (representative results from six independent experiments). 50 μm scale bar for low power fields and 20 μm scale bar for high power fields. e , f H&E staining and immunofluorescence staining of P2RX1 (green), CD66b (red) and DAPI (blue) in a total of 20 clinical PDAC liver metastasis samples were performed. Representative images are shown in ( e ), and the percentages of P2RX1-CD66b+ cells are shown in ( f ). 50 μm scale bar for low power fields and 20 μm scale bar for high power fields. g , h KPC cells were intrasplenically injected to seed livers of BM chimeras: WT → WT, P2rx1 −/− → P2rx1 −/− , WT → P2rx1 −/− , and P2rx1 −/− → WT. Neutrophils were depleted in WT → P2rx1 −/− and P2rx1 −/− → WT mice by intraperitoneal injection of anti-Ly6G (clone 1A8) antibody. At day 17, liver metastases were analyzed by in vivo imaging ( g , n = 5 for WT → WT, P2rx1 −/− → P2rx1 −/− and WT → P2rx1 −/− groups, n = 4 for WT → P2rx1 −/− + anti-Ly6G, P2rx1 −/− → WT and P2rx1 −/− → WT + anti-Ly6G groups, two independent experiments), and representative images of liver metastatic samples were shown ( h ). Bars represent mean ± standard deviation in ( f , g ). P values are derived from two-sided Student’s t test ( c , f ), or one-way ANOVA and Tukey’s multiple comparisons test ( g ). Source data are provided as a Source data file.
    Figure Legend Snippet: a Single cell suspension was obtained from mouse spleen and expression of P2RX1 was determined in indicated immune cell types ( n = 3 per group, two independent experiments). Left dotted line indicates the mean of negative control (NC) (secondary antibody only), and right dotted line indicates the mean of Ly6G+ cells. b KPC cells were intrasplenically injected to seed livers of WT mice. A single cell suspension was obtained from liver metastases and adjacent liver tissues of WT mice at day 17. The frequency of CD45+P2RX1+ cells was determined by flow cytometry ( n = 4 per group, three independent experiments). c KPC cells were intrasplenically injected to seed livers of WT mice. Immune cells were enriched from single cell suspension of liver metastases and adjacent liver tissues at day 17. P2RX1 expression in the indicated immune cell types was determined by flow cytometry ( n = 4 per group, three independent experiments). d Representative images of H&E staining and immunofluorescence staining of P2RX1 (Green), Ly6G (Red) and DAPI (Blue) in KPC mice spontaneous liver metastases (representative results from six independent experiments). 50 μm scale bar for low power fields and 20 μm scale bar for high power fields. e , f H&E staining and immunofluorescence staining of P2RX1 (green), CD66b (red) and DAPI (blue) in a total of 20 clinical PDAC liver metastasis samples were performed. Representative images are shown in ( e ), and the percentages of P2RX1-CD66b+ cells are shown in ( f ). 50 μm scale bar for low power fields and 20 μm scale bar for high power fields. g , h KPC cells were intrasplenically injected to seed livers of BM chimeras: WT → WT, P2rx1 −/− → P2rx1 −/− , WT → P2rx1 −/− , and P2rx1 −/− → WT. Neutrophils were depleted in WT → P2rx1 −/− and P2rx1 −/− → WT mice by intraperitoneal injection of anti-Ly6G (clone 1A8) antibody. At day 17, liver metastases were analyzed by in vivo imaging ( g , n = 5 for WT → WT, P2rx1 −/− → P2rx1 −/− and WT → P2rx1 −/− groups, n = 4 for WT → P2rx1 −/− + anti-Ly6G, P2rx1 −/− → WT and P2rx1 −/− → WT + anti-Ly6G groups, two independent experiments), and representative images of liver metastatic samples were shown ( h ). Bars represent mean ± standard deviation in ( f , g ). P values are derived from two-sided Student’s t test ( c , f ), or one-way ANOVA and Tukey’s multiple comparisons test ( g ). Source data are provided as a Source data file.

    Techniques Used: Expressing, Negative Control, Injection, Flow Cytometry, Staining, Immunofluorescence, In Vivo Imaging, Standard Deviation, Derivative Assay

    a – g KPC cell was intrasplenically injected to seed livers of WT mice. Bone marrow (BM) and peripheral blood (PB) were obtained at day 17. Frequencies of neutrophils, P2RX1− neutrophils, and CXCR4+ neutrophils were determined by flow cytometry and quantitative results were shown ( n = 4 per group, three independent experiments). h Bone marrow neutrophils were isolated from WT mice and stimulated with indicated stimulus. RNA-seq were performed and Log 2 (FPKM+0.001) value of purinergic receptors were shown ( n = 1 per group). Bars represent mean ± standard deviation in ( e – g ). P values are derived from two-sided Student’s t test ( e – g ). Source data are provided as a Source data file.
    Figure Legend Snippet: a – g KPC cell was intrasplenically injected to seed livers of WT mice. Bone marrow (BM) and peripheral blood (PB) were obtained at day 17. Frequencies of neutrophils, P2RX1− neutrophils, and CXCR4+ neutrophils were determined by flow cytometry and quantitative results were shown ( n = 4 per group, three independent experiments). h Bone marrow neutrophils were isolated from WT mice and stimulated with indicated stimulus. RNA-seq were performed and Log 2 (FPKM+0.001) value of purinergic receptors were shown ( n = 1 per group). Bars represent mean ± standard deviation in ( e – g ). P values are derived from two-sided Student’s t test ( e – g ). Source data are provided as a Source data file.

    Techniques Used: Injection, Flow Cytometry, Isolation, RNA Sequencing Assay, Standard Deviation, Derivative Assay

    a , b KPC cells were intrasplenically injected to seed livers of WT and P2rx1 −/− mice. A single cell suspension was obtained from liver metastases at day 17. Then, P2RX1+ neutrophils were purified from WT mice, and P2rx1 −/− neutrophils were purified from P2rx1 −/− mice for RNA sequencing. The results of KEGG analysis are shown in ( a ), and comparative expression of genes is shown in ( b ) ( n = 1 per group). c Bone marrow neutrophils were isolated from WT and P2rx1 −/− mice and stimulated with tumor conditioned medium (TCM). The ECAR and OCR were then measured by a Seahorse assay in ( c ), and PD-L1 and TNF-α were detected by RT-qPCR in ( d ) ( n = 4 per group, two independent experiments). Glc, glucose; O (ECAR), oligomycin; 2-DG, 2-deoxyglucose; O (OCR), oligomycin; F, FCCP (carbonyl cyanide 4-[trifluoromethoxy] phenylhydrazone); A & R, antimycin A and rotenone. Bars represent mean ± standard deviation in ( c , d ). P values are derived from two-sided Student’s t test ( d ). Source data are provided as a Source data file.
    Figure Legend Snippet: a , b KPC cells were intrasplenically injected to seed livers of WT and P2rx1 −/− mice. A single cell suspension was obtained from liver metastases at day 17. Then, P2RX1+ neutrophils were purified from WT mice, and P2rx1 −/− neutrophils were purified from P2rx1 −/− mice for RNA sequencing. The results of KEGG analysis are shown in ( a ), and comparative expression of genes is shown in ( b ) ( n = 1 per group). c Bone marrow neutrophils were isolated from WT and P2rx1 −/− mice and stimulated with tumor conditioned medium (TCM). The ECAR and OCR were then measured by a Seahorse assay in ( c ), and PD-L1 and TNF-α were detected by RT-qPCR in ( d ) ( n = 4 per group, two independent experiments). Glc, glucose; O (ECAR), oligomycin; 2-DG, 2-deoxyglucose; O (OCR), oligomycin; F, FCCP (carbonyl cyanide 4-[trifluoromethoxy] phenylhydrazone); A & R, antimycin A and rotenone. Bars represent mean ± standard deviation in ( c , d ). P values are derived from two-sided Student’s t test ( d ). Source data are provided as a Source data file.

    Techniques Used: Injection, Purification, RNA Sequencing Assay, Expressing, Isolation, Quantitative RT-PCR, Standard Deviation, Derivative Assay

    a Gene set enrichment analysis comparing RNA-seq data of P2RX1+ neutrophils and P2rx1 −/− neutrophils based on Nrf2 target genes. The p value and normalized enrichment score (NES) were shown. b , c Single-cell suspensions were obtained from liver metastases of WT and P2rx1 −/− mice at day 17. Intracellular Nrf2 was stained and detected by flow cytometry ( b ) or laser scanning confocal microscopy ( c ) in WT P2RX1+ neutrophils and P2rx1 −/− neutrophils ( n = 4 per group, three independent experiments). The scale bar is 2.5 μm. d Bone marrow neutrophils were isolated from WT and P2rx1 −/− mice and stimulated with LPS + IFN-γ in the presence or absence of a Nrf2 inhibitor. The ECAR was then measured by a Seahorse assay ( n = 4 per group, two independent experiments). Glc, glucose; O, oligomycin; 2-DG, 2-deoxyglucose. e Bone marrow neutrophils were isolated from WT and P2rx1 −/− mice and stimulated with IL-4 in the presence or absence of a Nrf2 inhibitor. The OCR was then measured by a Seahorse assay ( n = 4 per group, two independent experiments). O, oligomycin; F, FCCP (carbonyl cyanide 4-[trifluoromethoxy] phenylhydrazone); A & R, antimycin A and rotenone. f Bone marrow neutrophils were isolated from WT or P2rx1 −/− mice and stimulated with LPS + IFN-γ and an inhibitor Nrf2 inhibitor. IL-1β and TNF-α were determined by RT-qPCR ( n = 4 per group, three independent experiments). Bars represent mean ± standard deviation in ( d – f ). P values are derived from permutation test ( a ), two-sided Student’s t test ( f ). Source data are provided as a Source data file.
    Figure Legend Snippet: a Gene set enrichment analysis comparing RNA-seq data of P2RX1+ neutrophils and P2rx1 −/− neutrophils based on Nrf2 target genes. The p value and normalized enrichment score (NES) were shown. b , c Single-cell suspensions were obtained from liver metastases of WT and P2rx1 −/− mice at day 17. Intracellular Nrf2 was stained and detected by flow cytometry ( b ) or laser scanning confocal microscopy ( c ) in WT P2RX1+ neutrophils and P2rx1 −/− neutrophils ( n = 4 per group, three independent experiments). The scale bar is 2.5 μm. d Bone marrow neutrophils were isolated from WT and P2rx1 −/− mice and stimulated with LPS + IFN-γ in the presence or absence of a Nrf2 inhibitor. The ECAR was then measured by a Seahorse assay ( n = 4 per group, two independent experiments). Glc, glucose; O, oligomycin; 2-DG, 2-deoxyglucose. e Bone marrow neutrophils were isolated from WT and P2rx1 −/− mice and stimulated with IL-4 in the presence or absence of a Nrf2 inhibitor. The OCR was then measured by a Seahorse assay ( n = 4 per group, two independent experiments). O, oligomycin; F, FCCP (carbonyl cyanide 4-[trifluoromethoxy] phenylhydrazone); A & R, antimycin A and rotenone. f Bone marrow neutrophils were isolated from WT or P2rx1 −/− mice and stimulated with LPS + IFN-γ and an inhibitor Nrf2 inhibitor. IL-1β and TNF-α were determined by RT-qPCR ( n = 4 per group, three independent experiments). Bars represent mean ± standard deviation in ( d – f ). P values are derived from permutation test ( a ), two-sided Student’s t test ( f ). Source data are provided as a Source data file.

    Techniques Used: RNA Sequencing Assay, Staining, Flow Cytometry, Confocal Microscopy, Isolation, Quantitative RT-PCR, Standard Deviation, Derivative Assay

    a A single cell suspension was obtained from liver metastases of WT and P2rx1 −/− mice at day 17. Flow cytometry was performed to detect the frequency of CD8+PD-1+ (upper) and Ly6G+PD-L1 + or Ly6G+PD-L1− (lower) cells. P2RX1 expression was further determined in Ly6G+PD-L1+ and Ly6G+PD-L1− cells from WT mice ( n = 4 per group, three independent experiments). b Bone marrow neutrophils were isolated from WT and P2rx1 −/− mice and stimulated with tumor conditioned medium (TCM) or GM-CSF in the presence of a Nrf2 inhibitor or anti-GM-CSF neutralizing antibody. PD-L1 expression was detected by flow cytometry ( n = 4 per group, three independent experiments). c Integrative Genomics Viewer (IGV) was used to predict the two peaks that PD-L1 gene might be mediated by Nrf2 (upper). Bone marrow neutrophils were isolated from WT and P2rx1 −/− mice and stimulated with GM-CSF. Binding of PD-L1 gene by Nrf2 was detected by ChIP-PCR ( n = 3 per group, two independent experiments). d Bone marrow neutrophils were isolated from WT and P2rx1 −/− mice and stimulated with GM-CSF. Intracellular ROS was detected by flow cytometry ( n = 4 per group, three independent experiments). e Antigen activated CTLs was co-cultured with GM-CSF primed WT or P2rx1 −/− neutrophils. Cell proliferation was analyzed with CSFE staining in the presence or absence of anti-PD-1 neutralizing antibody ( n = 4 per group, three independent experiments). f KPC cells were transfected with empty lentiviral vector (KPC-LV) or OVA (KPC-OVA). Antigen-activated CTLs were co-cultured with KPC-LV or KPC-OVA cells in the presence of GM-CSF-primed WT or P2rx1 −/− neutrophils. Cytotoxicity was determined in the presence or absence of anti-PD-1 neutralizing antibody by counting the number of PI+ cells ( n = 4 per group, three independent experiments). Bars represent mean ± standard deviation in ( f ). P values are derived from one-way ANOVA and Tukey’s multiple comparisons test ( f ). Source data are provided as a Source data file.
    Figure Legend Snippet: a A single cell suspension was obtained from liver metastases of WT and P2rx1 −/− mice at day 17. Flow cytometry was performed to detect the frequency of CD8+PD-1+ (upper) and Ly6G+PD-L1 + or Ly6G+PD-L1− (lower) cells. P2RX1 expression was further determined in Ly6G+PD-L1+ and Ly6G+PD-L1− cells from WT mice ( n = 4 per group, three independent experiments). b Bone marrow neutrophils were isolated from WT and P2rx1 −/− mice and stimulated with tumor conditioned medium (TCM) or GM-CSF in the presence of a Nrf2 inhibitor or anti-GM-CSF neutralizing antibody. PD-L1 expression was detected by flow cytometry ( n = 4 per group, three independent experiments). c Integrative Genomics Viewer (IGV) was used to predict the two peaks that PD-L1 gene might be mediated by Nrf2 (upper). Bone marrow neutrophils were isolated from WT and P2rx1 −/− mice and stimulated with GM-CSF. Binding of PD-L1 gene by Nrf2 was detected by ChIP-PCR ( n = 3 per group, two independent experiments). d Bone marrow neutrophils were isolated from WT and P2rx1 −/− mice and stimulated with GM-CSF. Intracellular ROS was detected by flow cytometry ( n = 4 per group, three independent experiments). e Antigen activated CTLs was co-cultured with GM-CSF primed WT or P2rx1 −/− neutrophils. Cell proliferation was analyzed with CSFE staining in the presence or absence of anti-PD-1 neutralizing antibody ( n = 4 per group, three independent experiments). f KPC cells were transfected with empty lentiviral vector (KPC-LV) or OVA (KPC-OVA). Antigen-activated CTLs were co-cultured with KPC-LV or KPC-OVA cells in the presence of GM-CSF-primed WT or P2rx1 −/− neutrophils. Cytotoxicity was determined in the presence or absence of anti-PD-1 neutralizing antibody by counting the number of PI+ cells ( n = 4 per group, three independent experiments). Bars represent mean ± standard deviation in ( f ). P values are derived from one-way ANOVA and Tukey’s multiple comparisons test ( f ). Source data are provided as a Source data file.

    Techniques Used: Flow Cytometry, Expressing, Isolation, Binding Assay, Cell Culture, Staining, Transfection, Plasmid Preparation, Standard Deviation, Derivative Assay

    p2rx1 labeling  (Alomone Labs)


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    Alomone Labs p2rx1 labeling
    a Volcano plots of differential gene expression in 145 primary PDAC, 46 adjacent pancreases, 25 liver metastases and 27 adjacent livers. Red dots represent upregulated immune-related genes, and blue dots represent downregulated immune-related genes. b Immunome analyses of 26 infiltrating immune cell types in adjacent pancreas, primary PDAC, adjacent liver tissue and metastatic PDAC samples. c GO Biological Process analyses of differentially expressed genes between adjacent liver tissue and metastatic PDAC samples. d Expression analyses of <t>P2RX1</t> in the adjacent pancreas, primary PDAC, adjacent liver tissue and metastatic PDAC samples from the GSE71729 and Renji cohorts. e Correlation analyses between P2RX1 and immune checkpoint molecules in metastatic PDAC samples. Bars represent mean ± standard deviation in ( d ). * P < 0.05, ** P < 0.01, and *** P < 0.001, by one-way ANOVA and Tukey’s multiple comparisons test ( d left), or Student’s t test ( d right). Source data are provided as a Source data file.
    P2rx1 Labeling, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/p2rx1 labeling/product/Alomone Labs
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    p2rx1 labeling - by Bioz Stars, 2023-02
    93/100 stars

    Images

    1) Product Images from "Identification of a subset of immunosuppressive P2RX1-negative neutrophils in pancreatic cancer liver metastasis"

    Article Title: Identification of a subset of immunosuppressive P2RX1-negative neutrophils in pancreatic cancer liver metastasis

    Journal: Nature Communications

    doi: 10.1038/s41467-020-20447-y

    a Volcano plots of differential gene expression in 145 primary PDAC, 46 adjacent pancreases, 25 liver metastases and 27 adjacent livers. Red dots represent upregulated immune-related genes, and blue dots represent downregulated immune-related genes. b Immunome analyses of 26 infiltrating immune cell types in adjacent pancreas, primary PDAC, adjacent liver tissue and metastatic PDAC samples. c GO Biological Process analyses of differentially expressed genes between adjacent liver tissue and metastatic PDAC samples. d Expression analyses of P2RX1 in the adjacent pancreas, primary PDAC, adjacent liver tissue and metastatic PDAC samples from the GSE71729 and Renji cohorts. e Correlation analyses between P2RX1 and immune checkpoint molecules in metastatic PDAC samples. Bars represent mean ± standard deviation in ( d ). * P < 0.05, ** P < 0.01, and *** P < 0.001, by one-way ANOVA and Tukey’s multiple comparisons test ( d left), or Student’s t test ( d right). Source data are provided as a Source data file.
    Figure Legend Snippet: a Volcano plots of differential gene expression in 145 primary PDAC, 46 adjacent pancreases, 25 liver metastases and 27 adjacent livers. Red dots represent upregulated immune-related genes, and blue dots represent downregulated immune-related genes. b Immunome analyses of 26 infiltrating immune cell types in adjacent pancreas, primary PDAC, adjacent liver tissue and metastatic PDAC samples. c GO Biological Process analyses of differentially expressed genes between adjacent liver tissue and metastatic PDAC samples. d Expression analyses of P2RX1 in the adjacent pancreas, primary PDAC, adjacent liver tissue and metastatic PDAC samples from the GSE71729 and Renji cohorts. e Correlation analyses between P2RX1 and immune checkpoint molecules in metastatic PDAC samples. Bars represent mean ± standard deviation in ( d ). * P < 0.05, ** P < 0.01, and *** P < 0.001, by one-way ANOVA and Tukey’s multiple comparisons test ( d left), or Student’s t test ( d right). Source data are provided as a Source data file.

    Techniques Used: Expressing, Standard Deviation

    a , b P2rx1 −/− mice were generated using CRISPR/Cas9 system. Schematic diagram was shown in ( a ) and genotyping results were shown in ( b ) (representative result from three independent experiments). c , d KPC cells were intrasplenically injected to seed livers of WT and P2rx1 −/− mice, and in vivo imaging was performed at sequential times. Representative images are shown in ( c ), and quantitative results are shown in ( d ) ( n = 6 per group, three independent experiments). e Representative images of liver metastatic samples harvested at day 17. f Liver weight of liver metastatic samples was measured at day 17 ( n = 5 per group, two independent experiments). g Survival analysis of liver metastatic WT or P2rx1 −/− mice within a duration of 5 weeks ( n = 10 per group, two independent experiments). h Normal liver (D0) and two sequential stages (D3 and D17) of liver metastases in WT and P2rx1 −/− mice were harvested for RNA-seq, and PCA analyses were performed ( n = 3 for D0, n = 4 for D3 and D17). i Representative Ki67 immunohistochemical staining of WT and P2rx1 −/− liver metastases at day 17 ( n = 4 per group, two independent experiments). 100 μm of scale bar for low power fields, 25 μm of scale bar for high power fields. j Heatmap of immune checkpoint molecules in liver metastases of WT or P2rx1 −/− mice at D3 and D17 ( n = 4 per group). Bars represent mean ± standard deviation in ( d , f ). P values are derived from two-sided Student’s t test ( d , f ), or log-rank test ( g ). Source data are provided as a Source data file.
    Figure Legend Snippet: a , b P2rx1 −/− mice were generated using CRISPR/Cas9 system. Schematic diagram was shown in ( a ) and genotyping results were shown in ( b ) (representative result from three independent experiments). c , d KPC cells were intrasplenically injected to seed livers of WT and P2rx1 −/− mice, and in vivo imaging was performed at sequential times. Representative images are shown in ( c ), and quantitative results are shown in ( d ) ( n = 6 per group, three independent experiments). e Representative images of liver metastatic samples harvested at day 17. f Liver weight of liver metastatic samples was measured at day 17 ( n = 5 per group, two independent experiments). g Survival analysis of liver metastatic WT or P2rx1 −/− mice within a duration of 5 weeks ( n = 10 per group, two independent experiments). h Normal liver (D0) and two sequential stages (D3 and D17) of liver metastases in WT and P2rx1 −/− mice were harvested for RNA-seq, and PCA analyses were performed ( n = 3 for D0, n = 4 for D3 and D17). i Representative Ki67 immunohistochemical staining of WT and P2rx1 −/− liver metastases at day 17 ( n = 4 per group, two independent experiments). 100 μm of scale bar for low power fields, 25 μm of scale bar for high power fields. j Heatmap of immune checkpoint molecules in liver metastases of WT or P2rx1 −/− mice at D3 and D17 ( n = 4 per group). Bars represent mean ± standard deviation in ( d , f ). P values are derived from two-sided Student’s t test ( d , f ), or log-rank test ( g ). Source data are provided as a Source data file.

    Techniques Used: Generated, CRISPR, Injection, In Vivo Imaging, RNA Sequencing Assay, Immunohistochemical staining, Staining, Standard Deviation, Derivative Assay

    a Single cell suspension was obtained from mouse spleen and expression of P2RX1 was determined in indicated immune cell types ( n = 3 per group, two independent experiments). Left dotted line indicates the mean of negative control (NC) (secondary antibody only), and right dotted line indicates the mean of Ly6G+ cells. b KPC cells were intrasplenically injected to seed livers of WT mice. A single cell suspension was obtained from liver metastases and adjacent liver tissues of WT mice at day 17. The frequency of CD45+P2RX1+ cells was determined by flow cytometry ( n = 4 per group, three independent experiments). c KPC cells were intrasplenically injected to seed livers of WT mice. Immune cells were enriched from single cell suspension of liver metastases and adjacent liver tissues at day 17. P2RX1 expression in the indicated immune cell types was determined by flow cytometry ( n = 4 per group, three independent experiments). d Representative images of H&E staining and immunofluorescence staining of P2RX1 (Green), Ly6G (Red) and DAPI (Blue) in KPC mice spontaneous liver metastases (representative results from six independent experiments). 50 μm scale bar for low power fields and 20 μm scale bar for high power fields. e , f H&E staining and immunofluorescence staining of P2RX1 (green), CD66b (red) and DAPI (blue) in a total of 20 clinical PDAC liver metastasis samples were performed. Representative images are shown in ( e ), and the percentages of P2RX1-CD66b+ cells are shown in ( f ). 50 μm scale bar for low power fields and 20 μm scale bar for high power fields. g , h KPC cells were intrasplenically injected to seed livers of BM chimeras: WT → WT, P2rx1 −/− → P2rx1 −/− , WT → P2rx1 −/− , and P2rx1 −/− → WT. Neutrophils were depleted in WT → P2rx1 −/− and P2rx1 −/− → WT mice by intraperitoneal injection of anti-Ly6G (clone 1A8) antibody. At day 17, liver metastases were analyzed by in vivo imaging ( g , n = 5 for WT → WT, P2rx1 −/− → P2rx1 −/− and WT → P2rx1 −/− groups, n = 4 for WT → P2rx1 −/− + anti-Ly6G, P2rx1 −/− → WT and P2rx1 −/− → WT + anti-Ly6G groups, two independent experiments), and representative images of liver metastatic samples were shown ( h ). Bars represent mean ± standard deviation in ( f , g ). P values are derived from two-sided Student’s t test ( c , f ), or one-way ANOVA and Tukey’s multiple comparisons test ( g ). Source data are provided as a Source data file.
    Figure Legend Snippet: a Single cell suspension was obtained from mouse spleen and expression of P2RX1 was determined in indicated immune cell types ( n = 3 per group, two independent experiments). Left dotted line indicates the mean of negative control (NC) (secondary antibody only), and right dotted line indicates the mean of Ly6G+ cells. b KPC cells were intrasplenically injected to seed livers of WT mice. A single cell suspension was obtained from liver metastases and adjacent liver tissues of WT mice at day 17. The frequency of CD45+P2RX1+ cells was determined by flow cytometry ( n = 4 per group, three independent experiments). c KPC cells were intrasplenically injected to seed livers of WT mice. Immune cells were enriched from single cell suspension of liver metastases and adjacent liver tissues at day 17. P2RX1 expression in the indicated immune cell types was determined by flow cytometry ( n = 4 per group, three independent experiments). d Representative images of H&E staining and immunofluorescence staining of P2RX1 (Green), Ly6G (Red) and DAPI (Blue) in KPC mice spontaneous liver metastases (representative results from six independent experiments). 50 μm scale bar for low power fields and 20 μm scale bar for high power fields. e , f H&E staining and immunofluorescence staining of P2RX1 (green), CD66b (red) and DAPI (blue) in a total of 20 clinical PDAC liver metastasis samples were performed. Representative images are shown in ( e ), and the percentages of P2RX1-CD66b+ cells are shown in ( f ). 50 μm scale bar for low power fields and 20 μm scale bar for high power fields. g , h KPC cells were intrasplenically injected to seed livers of BM chimeras: WT → WT, P2rx1 −/− → P2rx1 −/− , WT → P2rx1 −/− , and P2rx1 −/− → WT. Neutrophils were depleted in WT → P2rx1 −/− and P2rx1 −/− → WT mice by intraperitoneal injection of anti-Ly6G (clone 1A8) antibody. At day 17, liver metastases were analyzed by in vivo imaging ( g , n = 5 for WT → WT, P2rx1 −/− → P2rx1 −/− and WT → P2rx1 −/− groups, n = 4 for WT → P2rx1 −/− + anti-Ly6G, P2rx1 −/− → WT and P2rx1 −/− → WT + anti-Ly6G groups, two independent experiments), and representative images of liver metastatic samples were shown ( h ). Bars represent mean ± standard deviation in ( f , g ). P values are derived from two-sided Student’s t test ( c , f ), or one-way ANOVA and Tukey’s multiple comparisons test ( g ). Source data are provided as a Source data file.

    Techniques Used: Expressing, Negative Control, Injection, Flow Cytometry, Staining, Immunofluorescence, In Vivo Imaging, Standard Deviation, Derivative Assay

    a – g KPC cell was intrasplenically injected to seed livers of WT mice. Bone marrow (BM) and peripheral blood (PB) were obtained at day 17. Frequencies of neutrophils, P2RX1− neutrophils, and CXCR4+ neutrophils were determined by flow cytometry and quantitative results were shown ( n = 4 per group, three independent experiments). h Bone marrow neutrophils were isolated from WT mice and stimulated with indicated stimulus. RNA-seq were performed and Log 2 (FPKM+0.001) value of purinergic receptors were shown ( n = 1 per group). Bars represent mean ± standard deviation in ( e – g ). P values are derived from two-sided Student’s t test ( e – g ). Source data are provided as a Source data file.
    Figure Legend Snippet: a – g KPC cell was intrasplenically injected to seed livers of WT mice. Bone marrow (BM) and peripheral blood (PB) were obtained at day 17. Frequencies of neutrophils, P2RX1− neutrophils, and CXCR4+ neutrophils were determined by flow cytometry and quantitative results were shown ( n = 4 per group, three independent experiments). h Bone marrow neutrophils were isolated from WT mice and stimulated with indicated stimulus. RNA-seq were performed and Log 2 (FPKM+0.001) value of purinergic receptors were shown ( n = 1 per group). Bars represent mean ± standard deviation in ( e – g ). P values are derived from two-sided Student’s t test ( e – g ). Source data are provided as a Source data file.

    Techniques Used: Injection, Flow Cytometry, Isolation, RNA Sequencing Assay, Standard Deviation, Derivative Assay

    a , b KPC cells were intrasplenically injected to seed livers of WT and P2rx1 −/− mice. A single cell suspension was obtained from liver metastases at day 17. Then, P2RX1+ neutrophils were purified from WT mice, and P2rx1 −/− neutrophils were purified from P2rx1 −/− mice for RNA sequencing. The results of KEGG analysis are shown in ( a ), and comparative expression of genes is shown in ( b ) ( n = 1 per group). c Bone marrow neutrophils were isolated from WT and P2rx1 −/− mice and stimulated with tumor conditioned medium (TCM). The ECAR and OCR were then measured by a Seahorse assay in ( c ), and PD-L1 and TNF-α were detected by RT-qPCR in ( d ) ( n = 4 per group, two independent experiments). Glc, glucose; O (ECAR), oligomycin; 2-DG, 2-deoxyglucose; O (OCR), oligomycin; F, FCCP (carbonyl cyanide 4-[trifluoromethoxy] phenylhydrazone); A & R, antimycin A and rotenone. Bars represent mean ± standard deviation in ( c , d ). P values are derived from two-sided Student’s t test ( d ). Source data are provided as a Source data file.
    Figure Legend Snippet: a , b KPC cells were intrasplenically injected to seed livers of WT and P2rx1 −/− mice. A single cell suspension was obtained from liver metastases at day 17. Then, P2RX1+ neutrophils were purified from WT mice, and P2rx1 −/− neutrophils were purified from P2rx1 −/− mice for RNA sequencing. The results of KEGG analysis are shown in ( a ), and comparative expression of genes is shown in ( b ) ( n = 1 per group). c Bone marrow neutrophils were isolated from WT and P2rx1 −/− mice and stimulated with tumor conditioned medium (TCM). The ECAR and OCR were then measured by a Seahorse assay in ( c ), and PD-L1 and TNF-α were detected by RT-qPCR in ( d ) ( n = 4 per group, two independent experiments). Glc, glucose; O (ECAR), oligomycin; 2-DG, 2-deoxyglucose; O (OCR), oligomycin; F, FCCP (carbonyl cyanide 4-[trifluoromethoxy] phenylhydrazone); A & R, antimycin A and rotenone. Bars represent mean ± standard deviation in ( c , d ). P values are derived from two-sided Student’s t test ( d ). Source data are provided as a Source data file.

    Techniques Used: Injection, Purification, RNA Sequencing Assay, Expressing, Isolation, Quantitative RT-PCR, Standard Deviation, Derivative Assay

    a Gene set enrichment analysis comparing RNA-seq data of P2RX1+ neutrophils and P2rx1 −/− neutrophils based on Nrf2 target genes. The p value and normalized enrichment score (NES) were shown. b , c Single-cell suspensions were obtained from liver metastases of WT and P2rx1 −/− mice at day 17. Intracellular Nrf2 was stained and detected by flow cytometry ( b ) or laser scanning confocal microscopy ( c ) in WT P2RX1+ neutrophils and P2rx1 −/− neutrophils ( n = 4 per group, three independent experiments). The scale bar is 2.5 μm. d Bone marrow neutrophils were isolated from WT and P2rx1 −/− mice and stimulated with LPS + IFN-γ in the presence or absence of a Nrf2 inhibitor. The ECAR was then measured by a Seahorse assay ( n = 4 per group, two independent experiments). Glc, glucose; O, oligomycin; 2-DG, 2-deoxyglucose. e Bone marrow neutrophils were isolated from WT and P2rx1 −/− mice and stimulated with IL-4 in the presence or absence of a Nrf2 inhibitor. The OCR was then measured by a Seahorse assay ( n = 4 per group, two independent experiments). O, oligomycin; F, FCCP (carbonyl cyanide 4-[trifluoromethoxy] phenylhydrazone); A & R, antimycin A and rotenone. f Bone marrow neutrophils were isolated from WT or P2rx1 −/− mice and stimulated with LPS + IFN-γ and an inhibitor Nrf2 inhibitor. IL-1β and TNF-α were determined by RT-qPCR ( n = 4 per group, three independent experiments). Bars represent mean ± standard deviation in ( d – f ). P values are derived from permutation test ( a ), two-sided Student’s t test ( f ). Source data are provided as a Source data file.
    Figure Legend Snippet: a Gene set enrichment analysis comparing RNA-seq data of P2RX1+ neutrophils and P2rx1 −/− neutrophils based on Nrf2 target genes. The p value and normalized enrichment score (NES) were shown. b , c Single-cell suspensions were obtained from liver metastases of WT and P2rx1 −/− mice at day 17. Intracellular Nrf2 was stained and detected by flow cytometry ( b ) or laser scanning confocal microscopy ( c ) in WT P2RX1+ neutrophils and P2rx1 −/− neutrophils ( n = 4 per group, three independent experiments). The scale bar is 2.5 μm. d Bone marrow neutrophils were isolated from WT and P2rx1 −/− mice and stimulated with LPS + IFN-γ in the presence or absence of a Nrf2 inhibitor. The ECAR was then measured by a Seahorse assay ( n = 4 per group, two independent experiments). Glc, glucose; O, oligomycin; 2-DG, 2-deoxyglucose. e Bone marrow neutrophils were isolated from WT and P2rx1 −/− mice and stimulated with IL-4 in the presence or absence of a Nrf2 inhibitor. The OCR was then measured by a Seahorse assay ( n = 4 per group, two independent experiments). O, oligomycin; F, FCCP (carbonyl cyanide 4-[trifluoromethoxy] phenylhydrazone); A & R, antimycin A and rotenone. f Bone marrow neutrophils were isolated from WT or P2rx1 −/− mice and stimulated with LPS + IFN-γ and an inhibitor Nrf2 inhibitor. IL-1β and TNF-α were determined by RT-qPCR ( n = 4 per group, three independent experiments). Bars represent mean ± standard deviation in ( d – f ). P values are derived from permutation test ( a ), two-sided Student’s t test ( f ). Source data are provided as a Source data file.

    Techniques Used: RNA Sequencing Assay, Staining, Flow Cytometry, Confocal Microscopy, Isolation, Quantitative RT-PCR, Standard Deviation, Derivative Assay

    a A single cell suspension was obtained from liver metastases of WT and P2rx1 −/− mice at day 17. Flow cytometry was performed to detect the frequency of CD8+PD-1+ (upper) and Ly6G+PD-L1 + or Ly6G+PD-L1− (lower) cells. P2RX1 expression was further determined in Ly6G+PD-L1+ and Ly6G+PD-L1− cells from WT mice ( n = 4 per group, three independent experiments). b Bone marrow neutrophils were isolated from WT and P2rx1 −/− mice and stimulated with tumor conditioned medium (TCM) or GM-CSF in the presence of a Nrf2 inhibitor or anti-GM-CSF neutralizing antibody. PD-L1 expression was detected by flow cytometry ( n = 4 per group, three independent experiments). c Integrative Genomics Viewer (IGV) was used to predict the two peaks that PD-L1 gene might be mediated by Nrf2 (upper). Bone marrow neutrophils were isolated from WT and P2rx1 −/− mice and stimulated with GM-CSF. Binding of PD-L1 gene by Nrf2 was detected by ChIP-PCR ( n = 3 per group, two independent experiments). d Bone marrow neutrophils were isolated from WT and P2rx1 −/− mice and stimulated with GM-CSF. Intracellular ROS was detected by flow cytometry ( n = 4 per group, three independent experiments). e Antigen activated CTLs was co-cultured with GM-CSF primed WT or P2rx1 −/− neutrophils. Cell proliferation was analyzed with CSFE staining in the presence or absence of anti-PD-1 neutralizing antibody ( n = 4 per group, three independent experiments). f KPC cells were transfected with empty lentiviral vector (KPC-LV) or OVA (KPC-OVA). Antigen-activated CTLs were co-cultured with KPC-LV or KPC-OVA cells in the presence of GM-CSF-primed WT or P2rx1 −/− neutrophils. Cytotoxicity was determined in the presence or absence of anti-PD-1 neutralizing antibody by counting the number of PI+ cells ( n = 4 per group, three independent experiments). Bars represent mean ± standard deviation in ( f ). P values are derived from one-way ANOVA and Tukey’s multiple comparisons test ( f ). Source data are provided as a Source data file.
    Figure Legend Snippet: a A single cell suspension was obtained from liver metastases of WT and P2rx1 −/− mice at day 17. Flow cytometry was performed to detect the frequency of CD8+PD-1+ (upper) and Ly6G+PD-L1 + or Ly6G+PD-L1− (lower) cells. P2RX1 expression was further determined in Ly6G+PD-L1+ and Ly6G+PD-L1− cells from WT mice ( n = 4 per group, three independent experiments). b Bone marrow neutrophils were isolated from WT and P2rx1 −/− mice and stimulated with tumor conditioned medium (TCM) or GM-CSF in the presence of a Nrf2 inhibitor or anti-GM-CSF neutralizing antibody. PD-L1 expression was detected by flow cytometry ( n = 4 per group, three independent experiments). c Integrative Genomics Viewer (IGV) was used to predict the two peaks that PD-L1 gene might be mediated by Nrf2 (upper). Bone marrow neutrophils were isolated from WT and P2rx1 −/− mice and stimulated with GM-CSF. Binding of PD-L1 gene by Nrf2 was detected by ChIP-PCR ( n = 3 per group, two independent experiments). d Bone marrow neutrophils were isolated from WT and P2rx1 −/− mice and stimulated with GM-CSF. Intracellular ROS was detected by flow cytometry ( n = 4 per group, three independent experiments). e Antigen activated CTLs was co-cultured with GM-CSF primed WT or P2rx1 −/− neutrophils. Cell proliferation was analyzed with CSFE staining in the presence or absence of anti-PD-1 neutralizing antibody ( n = 4 per group, three independent experiments). f KPC cells were transfected with empty lentiviral vector (KPC-LV) or OVA (KPC-OVA). Antigen-activated CTLs were co-cultured with KPC-LV or KPC-OVA cells in the presence of GM-CSF-primed WT or P2rx1 −/− neutrophils. Cytotoxicity was determined in the presence or absence of anti-PD-1 neutralizing antibody by counting the number of PI+ cells ( n = 4 per group, three independent experiments). Bars represent mean ± standard deviation in ( f ). P values are derived from one-way ANOVA and Tukey’s multiple comparisons test ( f ). Source data are provided as a Source data file.

    Techniques Used: Flow Cytometry, Expressing, Isolation, Binding Assay, Cell Culture, Staining, Transfection, Plasmid Preparation, Standard Deviation, Derivative Assay

    p2rx1 labeling  (Alomone Labs)


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    Alomone Labs p2rx1 labeling
    a Volcano plots of differential gene expression in 145 primary PDAC, 46 adjacent pancreases, 25 liver metastases and 27 adjacent livers. Red dots represent upregulated immune-related genes, and blue dots represent downregulated immune-related genes. b Immunome analyses of 26 infiltrating immune cell types in adjacent pancreas, primary PDAC, adjacent liver tissue and metastatic PDAC samples. c GO Biological Process analyses of differentially expressed genes between adjacent liver tissue and metastatic PDAC samples. d Expression analyses of <t>P2RX1</t> in the adjacent pancreas, primary PDAC, adjacent liver tissue and metastatic PDAC samples from the GSE71729 and Renji cohorts. e Correlation analyses between P2RX1 and immune checkpoint molecules in metastatic PDAC samples. Bars represent mean ± standard deviation in ( d ). * P < 0.05, ** P < 0.01, and *** P < 0.001, by one-way ANOVA and Tukey’s multiple comparisons test ( d left), or Student’s t test ( d right). Source data are provided as a Source data file.
    P2rx1 Labeling, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Identification of a subset of immunosuppressive P2RX1-negative neutrophils in pancreatic cancer liver metastasis"

    Article Title: Identification of a subset of immunosuppressive P2RX1-negative neutrophils in pancreatic cancer liver metastasis

    Journal: Nature Communications

    doi: 10.1038/s41467-020-20447-y

    a Volcano plots of differential gene expression in 145 primary PDAC, 46 adjacent pancreases, 25 liver metastases and 27 adjacent livers. Red dots represent upregulated immune-related genes, and blue dots represent downregulated immune-related genes. b Immunome analyses of 26 infiltrating immune cell types in adjacent pancreas, primary PDAC, adjacent liver tissue and metastatic PDAC samples. c GO Biological Process analyses of differentially expressed genes between adjacent liver tissue and metastatic PDAC samples. d Expression analyses of P2RX1 in the adjacent pancreas, primary PDAC, adjacent liver tissue and metastatic PDAC samples from the GSE71729 and Renji cohorts. e Correlation analyses between P2RX1 and immune checkpoint molecules in metastatic PDAC samples. Bars represent mean ± standard deviation in ( d ). * P < 0.05, ** P < 0.01, and *** P < 0.001, by one-way ANOVA and Tukey’s multiple comparisons test ( d left), or Student’s t test ( d right). Source data are provided as a Source data file.
    Figure Legend Snippet: a Volcano plots of differential gene expression in 145 primary PDAC, 46 adjacent pancreases, 25 liver metastases and 27 adjacent livers. Red dots represent upregulated immune-related genes, and blue dots represent downregulated immune-related genes. b Immunome analyses of 26 infiltrating immune cell types in adjacent pancreas, primary PDAC, adjacent liver tissue and metastatic PDAC samples. c GO Biological Process analyses of differentially expressed genes between adjacent liver tissue and metastatic PDAC samples. d Expression analyses of P2RX1 in the adjacent pancreas, primary PDAC, adjacent liver tissue and metastatic PDAC samples from the GSE71729 and Renji cohorts. e Correlation analyses between P2RX1 and immune checkpoint molecules in metastatic PDAC samples. Bars represent mean ± standard deviation in ( d ). * P < 0.05, ** P < 0.01, and *** P < 0.001, by one-way ANOVA and Tukey’s multiple comparisons test ( d left), or Student’s t test ( d right). Source data are provided as a Source data file.

    Techniques Used: Expressing, Standard Deviation

    a , b P2rx1 −/− mice were generated using CRISPR/Cas9 system. Schematic diagram was shown in ( a ) and genotyping results were shown in ( b ) (representative result from three independent experiments). c , d KPC cells were intrasplenically injected to seed livers of WT and P2rx1 −/− mice, and in vivo imaging was performed at sequential times. Representative images are shown in ( c ), and quantitative results are shown in ( d ) ( n = 6 per group, three independent experiments). e Representative images of liver metastatic samples harvested at day 17. f Liver weight of liver metastatic samples was measured at day 17 ( n = 5 per group, two independent experiments). g Survival analysis of liver metastatic WT or P2rx1 −/− mice within a duration of 5 weeks ( n = 10 per group, two independent experiments). h Normal liver (D0) and two sequential stages (D3 and D17) of liver metastases in WT and P2rx1 −/− mice were harvested for RNA-seq, and PCA analyses were performed ( n = 3 for D0, n = 4 for D3 and D17). i Representative Ki67 immunohistochemical staining of WT and P2rx1 −/− liver metastases at day 17 ( n = 4 per group, two independent experiments). 100 μm of scale bar for low power fields, 25 μm of scale bar for high power fields. j Heatmap of immune checkpoint molecules in liver metastases of WT or P2rx1 −/− mice at D3 and D17 ( n = 4 per group). Bars represent mean ± standard deviation in ( d , f ). P values are derived from two-sided Student’s t test ( d , f ), or log-rank test ( g ). Source data are provided as a Source data file.
    Figure Legend Snippet: a , b P2rx1 −/− mice were generated using CRISPR/Cas9 system. Schematic diagram was shown in ( a ) and genotyping results were shown in ( b ) (representative result from three independent experiments). c , d KPC cells were intrasplenically injected to seed livers of WT and P2rx1 −/− mice, and in vivo imaging was performed at sequential times. Representative images are shown in ( c ), and quantitative results are shown in ( d ) ( n = 6 per group, three independent experiments). e Representative images of liver metastatic samples harvested at day 17. f Liver weight of liver metastatic samples was measured at day 17 ( n = 5 per group, two independent experiments). g Survival analysis of liver metastatic WT or P2rx1 −/− mice within a duration of 5 weeks ( n = 10 per group, two independent experiments). h Normal liver (D0) and two sequential stages (D3 and D17) of liver metastases in WT and P2rx1 −/− mice were harvested for RNA-seq, and PCA analyses were performed ( n = 3 for D0, n = 4 for D3 and D17). i Representative Ki67 immunohistochemical staining of WT and P2rx1 −/− liver metastases at day 17 ( n = 4 per group, two independent experiments). 100 μm of scale bar for low power fields, 25 μm of scale bar for high power fields. j Heatmap of immune checkpoint molecules in liver metastases of WT or P2rx1 −/− mice at D3 and D17 ( n = 4 per group). Bars represent mean ± standard deviation in ( d , f ). P values are derived from two-sided Student’s t test ( d , f ), or log-rank test ( g ). Source data are provided as a Source data file.

    Techniques Used: Generated, CRISPR, Injection, In Vivo Imaging, RNA Sequencing Assay, Immunohistochemical staining, Staining, Standard Deviation, Derivative Assay

    a Single cell suspension was obtained from mouse spleen and expression of P2RX1 was determined in indicated immune cell types ( n = 3 per group, two independent experiments). Left dotted line indicates the mean of negative control (NC) (secondary antibody only), and right dotted line indicates the mean of Ly6G+ cells. b KPC cells were intrasplenically injected to seed livers of WT mice. A single cell suspension was obtained from liver metastases and adjacent liver tissues of WT mice at day 17. The frequency of CD45+P2RX1+ cells was determined by flow cytometry ( n = 4 per group, three independent experiments). c KPC cells were intrasplenically injected to seed livers of WT mice. Immune cells were enriched from single cell suspension of liver metastases and adjacent liver tissues at day 17. P2RX1 expression in the indicated immune cell types was determined by flow cytometry ( n = 4 per group, three independent experiments). d Representative images of H&E staining and immunofluorescence staining of P2RX1 (Green), Ly6G (Red) and DAPI (Blue) in KPC mice spontaneous liver metastases (representative results from six independent experiments). 50 μm scale bar for low power fields and 20 μm scale bar for high power fields. e , f H&E staining and immunofluorescence staining of P2RX1 (green), CD66b (red) and DAPI (blue) in a total of 20 clinical PDAC liver metastasis samples were performed. Representative images are shown in ( e ), and the percentages of P2RX1-CD66b+ cells are shown in ( f ). 50 μm scale bar for low power fields and 20 μm scale bar for high power fields. g , h KPC cells were intrasplenically injected to seed livers of BM chimeras: WT → WT, P2rx1 −/− → P2rx1 −/− , WT → P2rx1 −/− , and P2rx1 −/− → WT. Neutrophils were depleted in WT → P2rx1 −/− and P2rx1 −/− → WT mice by intraperitoneal injection of anti-Ly6G (clone 1A8) antibody. At day 17, liver metastases were analyzed by in vivo imaging ( g , n = 5 for WT → WT, P2rx1 −/− → P2rx1 −/− and WT → P2rx1 −/− groups, n = 4 for WT → P2rx1 −/− + anti-Ly6G, P2rx1 −/− → WT and P2rx1 −/− → WT + anti-Ly6G groups, two independent experiments), and representative images of liver metastatic samples were shown ( h ). Bars represent mean ± standard deviation in ( f , g ). P values are derived from two-sided Student’s t test ( c , f ), or one-way ANOVA and Tukey’s multiple comparisons test ( g ). Source data are provided as a Source data file.
    Figure Legend Snippet: a Single cell suspension was obtained from mouse spleen and expression of P2RX1 was determined in indicated immune cell types ( n = 3 per group, two independent experiments). Left dotted line indicates the mean of negative control (NC) (secondary antibody only), and right dotted line indicates the mean of Ly6G+ cells. b KPC cells were intrasplenically injected to seed livers of WT mice. A single cell suspension was obtained from liver metastases and adjacent liver tissues of WT mice at day 17. The frequency of CD45+P2RX1+ cells was determined by flow cytometry ( n = 4 per group, three independent experiments). c KPC cells were intrasplenically injected to seed livers of WT mice. Immune cells were enriched from single cell suspension of liver metastases and adjacent liver tissues at day 17. P2RX1 expression in the indicated immune cell types was determined by flow cytometry ( n = 4 per group, three independent experiments). d Representative images of H&E staining and immunofluorescence staining of P2RX1 (Green), Ly6G (Red) and DAPI (Blue) in KPC mice spontaneous liver metastases (representative results from six independent experiments). 50 μm scale bar for low power fields and 20 μm scale bar for high power fields. e , f H&E staining and immunofluorescence staining of P2RX1 (green), CD66b (red) and DAPI (blue) in a total of 20 clinical PDAC liver metastasis samples were performed. Representative images are shown in ( e ), and the percentages of P2RX1-CD66b+ cells are shown in ( f ). 50 μm scale bar for low power fields and 20 μm scale bar for high power fields. g , h KPC cells were intrasplenically injected to seed livers of BM chimeras: WT → WT, P2rx1 −/− → P2rx1 −/− , WT → P2rx1 −/− , and P2rx1 −/− → WT. Neutrophils were depleted in WT → P2rx1 −/− and P2rx1 −/− → WT mice by intraperitoneal injection of anti-Ly6G (clone 1A8) antibody. At day 17, liver metastases were analyzed by in vivo imaging ( g , n = 5 for WT → WT, P2rx1 −/− → P2rx1 −/− and WT → P2rx1 −/− groups, n = 4 for WT → P2rx1 −/− + anti-Ly6G, P2rx1 −/− → WT and P2rx1 −/− → WT + anti-Ly6G groups, two independent experiments), and representative images of liver metastatic samples were shown ( h ). Bars represent mean ± standard deviation in ( f , g ). P values are derived from two-sided Student’s t test ( c , f ), or one-way ANOVA and Tukey’s multiple comparisons test ( g ). Source data are provided as a Source data file.

    Techniques Used: Expressing, Negative Control, Injection, Flow Cytometry, Staining, Immunofluorescence, In Vivo Imaging, Standard Deviation, Derivative Assay

    a – g KPC cell was intrasplenically injected to seed livers of WT mice. Bone marrow (BM) and peripheral blood (PB) were obtained at day 17. Frequencies of neutrophils, P2RX1− neutrophils, and CXCR4+ neutrophils were determined by flow cytometry and quantitative results were shown ( n = 4 per group, three independent experiments). h Bone marrow neutrophils were isolated from WT mice and stimulated with indicated stimulus. RNA-seq were performed and Log 2 (FPKM+0.001) value of purinergic receptors were shown ( n = 1 per group). Bars represent mean ± standard deviation in ( e – g ). P values are derived from two-sided Student’s t test ( e – g ). Source data are provided as a Source data file.
    Figure Legend Snippet: a – g KPC cell was intrasplenically injected to seed livers of WT mice. Bone marrow (BM) and peripheral blood (PB) were obtained at day 17. Frequencies of neutrophils, P2RX1− neutrophils, and CXCR4+ neutrophils were determined by flow cytometry and quantitative results were shown ( n = 4 per group, three independent experiments). h Bone marrow neutrophils were isolated from WT mice and stimulated with indicated stimulus. RNA-seq were performed and Log 2 (FPKM+0.001) value of purinergic receptors were shown ( n = 1 per group). Bars represent mean ± standard deviation in ( e – g ). P values are derived from two-sided Student’s t test ( e – g ). Source data are provided as a Source data file.

    Techniques Used: Injection, Flow Cytometry, Isolation, RNA Sequencing Assay, Standard Deviation, Derivative Assay

    a , b KPC cells were intrasplenically injected to seed livers of WT and P2rx1 −/− mice. A single cell suspension was obtained from liver metastases at day 17. Then, P2RX1+ neutrophils were purified from WT mice, and P2rx1 −/− neutrophils were purified from P2rx1 −/− mice for RNA sequencing. The results of KEGG analysis are shown in ( a ), and comparative expression of genes is shown in ( b ) ( n = 1 per group). c Bone marrow neutrophils were isolated from WT and P2rx1 −/− mice and stimulated with tumor conditioned medium (TCM). The ECAR and OCR were then measured by a Seahorse assay in ( c ), and PD-L1 and TNF-α were detected by RT-qPCR in ( d ) ( n = 4 per group, two independent experiments). Glc, glucose; O (ECAR), oligomycin; 2-DG, 2-deoxyglucose; O (OCR), oligomycin; F, FCCP (carbonyl cyanide 4-[trifluoromethoxy] phenylhydrazone); A & R, antimycin A and rotenone. Bars represent mean ± standard deviation in ( c , d ). P values are derived from two-sided Student’s t test ( d ). Source data are provided as a Source data file.
    Figure Legend Snippet: a , b KPC cells were intrasplenically injected to seed livers of WT and P2rx1 −/− mice. A single cell suspension was obtained from liver metastases at day 17. Then, P2RX1+ neutrophils were purified from WT mice, and P2rx1 −/− neutrophils were purified from P2rx1 −/− mice for RNA sequencing. The results of KEGG analysis are shown in ( a ), and comparative expression of genes is shown in ( b ) ( n = 1 per group). c Bone marrow neutrophils were isolated from WT and P2rx1 −/− mice and stimulated with tumor conditioned medium (TCM). The ECAR and OCR were then measured by a Seahorse assay in ( c ), and PD-L1 and TNF-α were detected by RT-qPCR in ( d ) ( n = 4 per group, two independent experiments). Glc, glucose; O (ECAR), oligomycin; 2-DG, 2-deoxyglucose; O (OCR), oligomycin; F, FCCP (carbonyl cyanide 4-[trifluoromethoxy] phenylhydrazone); A & R, antimycin A and rotenone. Bars represent mean ± standard deviation in ( c , d ). P values are derived from two-sided Student’s t test ( d ). Source data are provided as a Source data file.

    Techniques Used: Injection, Purification, RNA Sequencing Assay, Expressing, Isolation, Quantitative RT-PCR, Standard Deviation, Derivative Assay

    a Gene set enrichment analysis comparing RNA-seq data of P2RX1+ neutrophils and P2rx1 −/− neutrophils based on Nrf2 target genes. The p value and normalized enrichment score (NES) were shown. b , c Single-cell suspensions were obtained from liver metastases of WT and P2rx1 −/− mice at day 17. Intracellular Nrf2 was stained and detected by flow cytometry ( b ) or laser scanning confocal microscopy ( c ) in WT P2RX1+ neutrophils and P2rx1 −/− neutrophils ( n = 4 per group, three independent experiments). The scale bar is 2.5 μm. d Bone marrow neutrophils were isolated from WT and P2rx1 −/− mice and stimulated with LPS + IFN-γ in the presence or absence of a Nrf2 inhibitor. The ECAR was then measured by a Seahorse assay ( n = 4 per group, two independent experiments). Glc, glucose; O, oligomycin; 2-DG, 2-deoxyglucose. e Bone marrow neutrophils were isolated from WT and P2rx1 −/− mice and stimulated with IL-4 in the presence or absence of a Nrf2 inhibitor. The OCR was then measured by a Seahorse assay ( n = 4 per group, two independent experiments). O, oligomycin; F, FCCP (carbonyl cyanide 4-[trifluoromethoxy] phenylhydrazone); A & R, antimycin A and rotenone. f Bone marrow neutrophils were isolated from WT or P2rx1 −/− mice and stimulated with LPS + IFN-γ and an inhibitor Nrf2 inhibitor. IL-1β and TNF-α were determined by RT-qPCR ( n = 4 per group, three independent experiments). Bars represent mean ± standard deviation in ( d – f ). P values are derived from permutation test ( a ), two-sided Student’s t test ( f ). Source data are provided as a Source data file.
    Figure Legend Snippet: a Gene set enrichment analysis comparing RNA-seq data of P2RX1+ neutrophils and P2rx1 −/− neutrophils based on Nrf2 target genes. The p value and normalized enrichment score (NES) were shown. b , c Single-cell suspensions were obtained from liver metastases of WT and P2rx1 −/− mice at day 17. Intracellular Nrf2 was stained and detected by flow cytometry ( b ) or laser scanning confocal microscopy ( c ) in WT P2RX1+ neutrophils and P2rx1 −/− neutrophils ( n = 4 per group, three independent experiments). The scale bar is 2.5 μm. d Bone marrow neutrophils were isolated from WT and P2rx1 −/− mice and stimulated with LPS + IFN-γ in the presence or absence of a Nrf2 inhibitor. The ECAR was then measured by a Seahorse assay ( n = 4 per group, two independent experiments). Glc, glucose; O, oligomycin; 2-DG, 2-deoxyglucose. e Bone marrow neutrophils were isolated from WT and P2rx1 −/− mice and stimulated with IL-4 in the presence or absence of a Nrf2 inhibitor. The OCR was then measured by a Seahorse assay ( n = 4 per group, two independent experiments). O, oligomycin; F, FCCP (carbonyl cyanide 4-[trifluoromethoxy] phenylhydrazone); A & R, antimycin A and rotenone. f Bone marrow neutrophils were isolated from WT or P2rx1 −/− mice and stimulated with LPS + IFN-γ and an inhibitor Nrf2 inhibitor. IL-1β and TNF-α were determined by RT-qPCR ( n = 4 per group, three independent experiments). Bars represent mean ± standard deviation in ( d – f ). P values are derived from permutation test ( a ), two-sided Student’s t test ( f ). Source data are provided as a Source data file.

    Techniques Used: RNA Sequencing Assay, Staining, Flow Cytometry, Confocal Microscopy, Isolation, Quantitative RT-PCR, Standard Deviation, Derivative Assay

    a A single cell suspension was obtained from liver metastases of WT and P2rx1 −/− mice at day 17. Flow cytometry was performed to detect the frequency of CD8+PD-1+ (upper) and Ly6G+PD-L1 + or Ly6G+PD-L1− (lower) cells. P2RX1 expression was further determined in Ly6G+PD-L1+ and Ly6G+PD-L1− cells from WT mice ( n = 4 per group, three independent experiments). b Bone marrow neutrophils were isolated from WT and P2rx1 −/− mice and stimulated with tumor conditioned medium (TCM) or GM-CSF in the presence of a Nrf2 inhibitor or anti-GM-CSF neutralizing antibody. PD-L1 expression was detected by flow cytometry ( n = 4 per group, three independent experiments). c Integrative Genomics Viewer (IGV) was used to predict the two peaks that PD-L1 gene might be mediated by Nrf2 (upper). Bone marrow neutrophils were isolated from WT and P2rx1 −/− mice and stimulated with GM-CSF. Binding of PD-L1 gene by Nrf2 was detected by ChIP-PCR ( n = 3 per group, two independent experiments). d Bone marrow neutrophils were isolated from WT and P2rx1 −/− mice and stimulated with GM-CSF. Intracellular ROS was detected by flow cytometry ( n = 4 per group, three independent experiments). e Antigen activated CTLs was co-cultured with GM-CSF primed WT or P2rx1 −/− neutrophils. Cell proliferation was analyzed with CSFE staining in the presence or absence of anti-PD-1 neutralizing antibody ( n = 4 per group, three independent experiments). f KPC cells were transfected with empty lentiviral vector (KPC-LV) or OVA (KPC-OVA). Antigen-activated CTLs were co-cultured with KPC-LV or KPC-OVA cells in the presence of GM-CSF-primed WT or P2rx1 −/− neutrophils. Cytotoxicity was determined in the presence or absence of anti-PD-1 neutralizing antibody by counting the number of PI+ cells ( n = 4 per group, three independent experiments). Bars represent mean ± standard deviation in ( f ). P values are derived from one-way ANOVA and Tukey’s multiple comparisons test ( f ). Source data are provided as a Source data file.
    Figure Legend Snippet: a A single cell suspension was obtained from liver metastases of WT and P2rx1 −/− mice at day 17. Flow cytometry was performed to detect the frequency of CD8+PD-1+ (upper) and Ly6G+PD-L1 + or Ly6G+PD-L1− (lower) cells. P2RX1 expression was further determined in Ly6G+PD-L1+ and Ly6G+PD-L1− cells from WT mice ( n = 4 per group, three independent experiments). b Bone marrow neutrophils were isolated from WT and P2rx1 −/− mice and stimulated with tumor conditioned medium (TCM) or GM-CSF in the presence of a Nrf2 inhibitor or anti-GM-CSF neutralizing antibody. PD-L1 expression was detected by flow cytometry ( n = 4 per group, three independent experiments). c Integrative Genomics Viewer (IGV) was used to predict the two peaks that PD-L1 gene might be mediated by Nrf2 (upper). Bone marrow neutrophils were isolated from WT and P2rx1 −/− mice and stimulated with GM-CSF. Binding of PD-L1 gene by Nrf2 was detected by ChIP-PCR ( n = 3 per group, two independent experiments). d Bone marrow neutrophils were isolated from WT and P2rx1 −/− mice and stimulated with GM-CSF. Intracellular ROS was detected by flow cytometry ( n = 4 per group, three independent experiments). e Antigen activated CTLs was co-cultured with GM-CSF primed WT or P2rx1 −/− neutrophils. Cell proliferation was analyzed with CSFE staining in the presence or absence of anti-PD-1 neutralizing antibody ( n = 4 per group, three independent experiments). f KPC cells were transfected with empty lentiviral vector (KPC-LV) or OVA (KPC-OVA). Antigen-activated CTLs were co-cultured with KPC-LV or KPC-OVA cells in the presence of GM-CSF-primed WT or P2rx1 −/− neutrophils. Cytotoxicity was determined in the presence or absence of anti-PD-1 neutralizing antibody by counting the number of PI+ cells ( n = 4 per group, three independent experiments). Bars represent mean ± standard deviation in ( f ). P values are derived from one-way ANOVA and Tukey’s multiple comparisons test ( f ). Source data are provided as a Source data file.

    Techniques Used: Flow Cytometry, Expressing, Isolation, Binding Assay, Cell Culture, Staining, Transfection, Plasmid Preparation, Standard Deviation, Derivative Assay

    anti p2x1 antibodies  (Alomone Labs)


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    Alomone Labs anti p2x1 antibodies
    Expression and function of purinergic receptors. The mean fluorescence intensities (MFI) of basal or 10 μM TRAP-6-stimulated receptor expression, detected by flow cytometry, are shown for P2Y1 ( A ), P2Y12 ( B ) and <t>P2X1</t> ( C ). The function of the P2Y1 ( D ) and of the P2X1 ( E ) receptor was measured by calcium-induced fluorescence after selective stimulation, shown as mean values in relative fluorescence units (RFU). The function of the P2Y12 receptor ( F ) is expressed as platelet reactivity index (PRI) determined by the flow cytometric VASP assay. Mean ± SEM; n = 6; * p < 0.05 (for RT-APC in black, for C-APC in blue). C-APC, cold-stored apheresis-derived platelet concentrates; PB, peripheral blood; RT, room temperature; SEM, standard error of mean; TRAP-6, thrombin receptor activating peptide; VASP, vasodilator-stimulated phosphoprotein.
    Anti P2x1 Antibodies, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "The Impact of Cold Storage on Adenosine Diphosphate-Mediated Platelet Responsiveness"

    Article Title: The Impact of Cold Storage on Adenosine Diphosphate-Mediated Platelet Responsiveness

    Journal: TH Open: Companion Journal to Thrombosis and Haemostasis

    doi: 10.1055/s-0040-1714254

    Expression and function of purinergic receptors. The mean fluorescence intensities (MFI) of basal or 10 μM TRAP-6-stimulated receptor expression, detected by flow cytometry, are shown for P2Y1 ( A ), P2Y12 ( B ) and P2X1 ( C ). The function of the P2Y1 ( D ) and of the P2X1 ( E ) receptor was measured by calcium-induced fluorescence after selective stimulation, shown as mean values in relative fluorescence units (RFU). The function of the P2Y12 receptor ( F ) is expressed as platelet reactivity index (PRI) determined by the flow cytometric VASP assay. Mean ± SEM; n = 6; * p < 0.05 (for RT-APC in black, for C-APC in blue). C-APC, cold-stored apheresis-derived platelet concentrates; PB, peripheral blood; RT, room temperature; SEM, standard error of mean; TRAP-6, thrombin receptor activating peptide; VASP, vasodilator-stimulated phosphoprotein.
    Figure Legend Snippet: Expression and function of purinergic receptors. The mean fluorescence intensities (MFI) of basal or 10 μM TRAP-6-stimulated receptor expression, detected by flow cytometry, are shown for P2Y1 ( A ), P2Y12 ( B ) and P2X1 ( C ). The function of the P2Y1 ( D ) and of the P2X1 ( E ) receptor was measured by calcium-induced fluorescence after selective stimulation, shown as mean values in relative fluorescence units (RFU). The function of the P2Y12 receptor ( F ) is expressed as platelet reactivity index (PRI) determined by the flow cytometric VASP assay. Mean ± SEM; n = 6; * p < 0.05 (for RT-APC in black, for C-APC in blue). C-APC, cold-stored apheresis-derived platelet concentrates; PB, peripheral blood; RT, room temperature; SEM, standard error of mean; TRAP-6, thrombin receptor activating peptide; VASP, vasodilator-stimulated phosphoprotein.

    Techniques Used: Expressing, Fluorescence, Flow Cytometry, Derivative Assay

    anti p2x1  (Alomone Labs)


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    Alomone Labs anti p2x1
    Anti P2x1, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 93 stars, based on 1 article reviews
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    anti p2x1 antibodies  (Alomone Labs)


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    Alomone Labs anti p2x1 antibodies
    The histograms show the mean fluorescence intensities (MFI) of basal (white columns) and 10 μM TRAP-6 stimulated (shaded columns) P2Y1- (A), P2Y12- (B), and <t>P2X1-</t> (C) receptor expression in fresh PB samples and in stored WB units at different time points as indicated. Results are presented in absolute arbitrary units (AU) as mean fluorescence ± SEM; n = 6; *: p < 0.05 (compared to basal values).
    Anti P2x1 Antibodies, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti p2x1 antibodies/product/Alomone Labs
    Average 93 stars, based on 1 article reviews
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    1) Product Images from "The role of adenosine diphosphate mediated platelet responsiveness for the stability of platelet integrity in citrated whole blood under ex vivo conditions"

    Article Title: The role of adenosine diphosphate mediated platelet responsiveness for the stability of platelet integrity in citrated whole blood under ex vivo conditions

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0188193

    The histograms show the mean fluorescence intensities (MFI) of basal (white columns) and 10 μM TRAP-6 stimulated (shaded columns) P2Y1- (A), P2Y12- (B), and P2X1- (C) receptor expression in fresh PB samples and in stored WB units at different time points as indicated. Results are presented in absolute arbitrary units (AU) as mean fluorescence ± SEM; n = 6; *: p < 0.05 (compared to basal values).
    Figure Legend Snippet: The histograms show the mean fluorescence intensities (MFI) of basal (white columns) and 10 μM TRAP-6 stimulated (shaded columns) P2Y1- (A), P2Y12- (B), and P2X1- (C) receptor expression in fresh PB samples and in stored WB units at different time points as indicated. Results are presented in absolute arbitrary units (AU) as mean fluorescence ± SEM; n = 6; *: p < 0.05 (compared to basal values).

    Techniques Used: Fluorescence, Expressing

    Calcium induced fluorescence curves were generated with Fluo-4AM loaded freshly washed platelets from PB samples (black line) and from WB units on Day 0 (dashed line), on Day 2 (dotted line) and on Day 5 (gray line) after stimulation with the P2Y1 agonist MRS2365 (A) or the P2X1 agonist α,β-MeATP (C). Mean fluorescence curves are presented (expressed in relative fluorescence units, R.F.U.); n = 6. For the P2Y12 receptor activity, mean PRI values (B) were determined in PB samples and in stored WB units at different time points as indicated. Results are presented as mean % (PRI) ± SEM; n = 4.
    Figure Legend Snippet: Calcium induced fluorescence curves were generated with Fluo-4AM loaded freshly washed platelets from PB samples (black line) and from WB units on Day 0 (dashed line), on Day 2 (dotted line) and on Day 5 (gray line) after stimulation with the P2Y1 agonist MRS2365 (A) or the P2X1 agonist α,β-MeATP (C). Mean fluorescence curves are presented (expressed in relative fluorescence units, R.F.U.); n = 6. For the P2Y12 receptor activity, mean PRI values (B) were determined in PB samples and in stored WB units at different time points as indicated. Results are presented as mean % (PRI) ± SEM; n = 4.

    Techniques Used: Fluorescence, Generated, Activity Assay

    rabbit polyclonal anti p2x 7  (Alomone Labs)


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    Alomone Labs rabbit polyclonal anti p2x 7
    A, Relative expression of <t>P2X</t> <t>7</t> mRNA detected by qPCR. B, SDS-PAGE electrophoresis of P2X 7 receptor protein. C, Relative expression of P2X 7 receptor protein; ** P < 0.01, versus Ctrl group; n = 5.
    Rabbit Polyclonal Anti P2x 7, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Effect of naringin on gp120-induced injury mediated by P2X 7 receptors in rat primary cultured microglia"

    Article Title: Effect of naringin on gp120-induced injury mediated by P2X 7 receptors in rat primary cultured microglia

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0183688

    A, Relative expression of P2X 7 mRNA detected by qPCR. B, SDS-PAGE electrophoresis of P2X 7 receptor protein. C, Relative expression of P2X 7 receptor protein; ** P < 0.01, versus Ctrl group; n = 5.
    Figure Legend Snippet: A, Relative expression of P2X 7 mRNA detected by qPCR. B, SDS-PAGE electrophoresis of P2X 7 receptor protein. C, Relative expression of P2X 7 receptor protein; ** P < 0.01, versus Ctrl group; n = 5.

    Techniques Used: Expressing, SDS Page, Electrophoresis

    A, SDS-PAGE electrophoresis of P2X 7 receptor proteins in primary cultured microglia. B, Relative expression level of P2X 7 receptor protein in primary cultured microglia; ** P < 0.01, versus Ctrl group; ## P < 0.01, versus gp120 group; n = 5.
    Figure Legend Snippet: A, SDS-PAGE electrophoresis of P2X 7 receptor proteins in primary cultured microglia. B, Relative expression level of P2X 7 receptor protein in primary cultured microglia; ** P < 0.01, versus Ctrl group; ## P < 0.01, versus gp120 group; n = 5.

    Techniques Used: SDS Page, Electrophoresis, Cell Culture, Expressing

    A, Typical fluorescence image of primary cultured microglia; green signal represents CD11b staining with FITC; red signal indicates P2X 7 staining with TRITC; Merge represents the P2X 7 and CD11b double staining image; arrows represent microglia with co-expression of P2X 7 receptor and CD11b. B, Co-expression values of CD11b and P2X 7 receptor in each group; ** P < 0.01, versus Ctrl group; ## P < 0.01, versus gp120 group; n = 3.
    Figure Legend Snippet: A, Typical fluorescence image of primary cultured microglia; green signal represents CD11b staining with FITC; red signal indicates P2X 7 staining with TRITC; Merge represents the P2X 7 and CD11b double staining image; arrows represent microglia with co-expression of P2X 7 receptor and CD11b. B, Co-expression values of CD11b and P2X 7 receptor in each group; ** P < 0.01, versus Ctrl group; ## P < 0.01, versus gp120 group; n = 3.

    Techniques Used: Fluorescence, Cell Culture, Staining, Double Staining, Expressing

    anti p2x1 antibodies  (Alomone Labs)


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    Alomone Labs anti p2x1 antibodies
    Impaired calcium flux mediated by P2Y1 and <t>P2X1</t> receptors in platelets from stored APC.
    Anti P2x1 Antibodies, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti p2x1 antibodies/product/Alomone Labs
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    1) Product Images from "Expression and function of purinergic receptors in platelets from apheresis-derived platelet concentrates"

    Article Title: Expression and function of purinergic receptors in platelets from apheresis-derived platelet concentrates

    Journal: Blood Transfusion

    doi: 10.2450/2015.0073-15

    Impaired calcium flux mediated by P2Y1 and P2X1 receptors in platelets from stored APC.
    Figure Legend Snippet: Impaired calcium flux mediated by P2Y1 and P2X1 receptors in platelets from stored APC.

    Techniques Used:

    anti p2x1 antibodies  (Alomone Labs)


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    Alomone Labs anti p2x1 antibodies
    Washed platelets (3 x 10 8 per mL resting in HEPES buffer at RT) and platelets in PRP were stained with purinergic receptor specific antibodies at different time points after preparation as indicated. The basal receptor surface expression was quantified by flow cytometry. The histograms show the mean fluorescence intensities (MFI) of P2Y1 (A and D), P2Y12 (B and E) and <t>P2X1</t> (C and F). Results are presented in absolute arbitrary units as mean ± SEM; n = 5; *: p < 0.05 (compared to 0 min).
    Anti P2x1 Antibodies, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti p2x1 antibodies/product/Alomone Labs
    Average 93 stars, based on 1 article reviews
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    anti p2x1 antibodies - by Bioz Stars, 2023-02
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    1) Product Images from "Role of Purinergic Receptor Expression and Function for Reduced Responsiveness to Adenosine Diphosphate in Washed Human Platelets"

    Article Title: Role of Purinergic Receptor Expression and Function for Reduced Responsiveness to Adenosine Diphosphate in Washed Human Platelets

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0147370

    Washed platelets (3 x 10 8 per mL resting in HEPES buffer at RT) and platelets in PRP were stained with purinergic receptor specific antibodies at different time points after preparation as indicated. The basal receptor surface expression was quantified by flow cytometry. The histograms show the mean fluorescence intensities (MFI) of P2Y1 (A and D), P2Y12 (B and E) and P2X1 (C and F). Results are presented in absolute arbitrary units as mean ± SEM; n = 5; *: p < 0.05 (compared to 0 min).
    Figure Legend Snippet: Washed platelets (3 x 10 8 per mL resting in HEPES buffer at RT) and platelets in PRP were stained with purinergic receptor specific antibodies at different time points after preparation as indicated. The basal receptor surface expression was quantified by flow cytometry. The histograms show the mean fluorescence intensities (MFI) of P2Y1 (A and D), P2Y12 (B and E) and P2X1 (C and F). Results are presented in absolute arbitrary units as mean ± SEM; n = 5; *: p < 0.05 (compared to 0 min).

    Techniques Used: Staining, Expressing, Flow Cytometry, Fluorescence

    The figures show the calcium induced fluorescence curves in Fluo-4AM loaded washed platelets (A) and in platelets from PRP (B) after stimulation with the P2X1 agonist α,β-MeATP at different time points after preparation as indicated. Mean fluorescence curves of five independent experiments are presented (relative fluorescence units, R.F.U.).
    Figure Legend Snippet: The figures show the calcium induced fluorescence curves in Fluo-4AM loaded washed platelets (A) and in platelets from PRP (B) after stimulation with the P2X1 agonist α,β-MeATP at different time points after preparation as indicated. Mean fluorescence curves of five independent experiments are presented (relative fluorescence units, R.F.U.).

    Techniques Used: Fluorescence

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    a Volcano plots of differential gene expression in 145 primary PDAC, 46 adjacent pancreases, 25 liver metastases and 27 adjacent livers. Red dots represent upregulated immune-related genes, and blue dots represent downregulated immune-related genes. b Immunome analyses of 26 infiltrating immune cell types in adjacent pancreas, primary PDAC, adjacent liver tissue and metastatic PDAC samples. c GO Biological Process analyses of differentially expressed genes between adjacent liver tissue and metastatic PDAC samples. d Expression analyses of <t>P2RX1</t> in the adjacent pancreas, primary PDAC, adjacent liver tissue and metastatic PDAC samples from the GSE71729 and Renji cohorts. e Correlation analyses between P2RX1 and immune checkpoint molecules in metastatic PDAC samples. Bars represent mean ± standard deviation in ( d ). * P < 0.05, ** P < 0.01, and *** P < 0.001, by one-way ANOVA and Tukey’s multiple comparisons test ( d left), or Student’s t test ( d right). Source data are provided as a Source data file.
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    a Volcano plots of differential gene expression in 145 primary PDAC, 46 adjacent pancreases, 25 liver metastases and 27 adjacent livers. Red dots represent upregulated immune-related genes, and blue dots represent downregulated immune-related genes. b Immunome analyses of 26 infiltrating immune cell types in adjacent pancreas, primary PDAC, adjacent liver tissue and metastatic PDAC samples. c GO Biological Process analyses of differentially expressed genes between adjacent liver tissue and metastatic PDAC samples. d Expression analyses of <t>P2RX1</t> in the adjacent pancreas, primary PDAC, adjacent liver tissue and metastatic PDAC samples from the GSE71729 and Renji cohorts. e Correlation analyses between P2RX1 and immune checkpoint molecules in metastatic PDAC samples. Bars represent mean ± standard deviation in ( d ). * P < 0.05, ** P < 0.01, and *** P < 0.001, by one-way ANOVA and Tukey’s multiple comparisons test ( d left), or Student’s t test ( d right). Source data are provided as a Source data file.
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    Expression and function of purinergic receptors. The mean fluorescence intensities (MFI) of basal or 10 μM TRAP-6-stimulated receptor expression, detected by flow cytometry, are shown for P2Y1 ( A ), P2Y12 ( B ) and <t>P2X1</t> ( C ). The function of the P2Y1 ( D ) and of the P2X1 ( E ) receptor was measured by calcium-induced fluorescence after selective stimulation, shown as mean values in relative fluorescence units (RFU). The function of the P2Y12 receptor ( F ) is expressed as platelet reactivity index (PRI) determined by the flow cytometric VASP assay. Mean ± SEM; n = 6; * p < 0.05 (for RT-APC in black, for C-APC in blue). C-APC, cold-stored apheresis-derived platelet concentrates; PB, peripheral blood; RT, room temperature; SEM, standard error of mean; TRAP-6, thrombin receptor activating peptide; VASP, vasodilator-stimulated phosphoprotein.
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    a Volcano plots of differential gene expression in 145 primary PDAC, 46 adjacent pancreases, 25 liver metastases and 27 adjacent livers. Red dots represent upregulated immune-related genes, and blue dots represent downregulated immune-related genes. b Immunome analyses of 26 infiltrating immune cell types in adjacent pancreas, primary PDAC, adjacent liver tissue and metastatic PDAC samples. c GO Biological Process analyses of differentially expressed genes between adjacent liver tissue and metastatic PDAC samples. d Expression analyses of P2RX1 in the adjacent pancreas, primary PDAC, adjacent liver tissue and metastatic PDAC samples from the GSE71729 and Renji cohorts. e Correlation analyses between P2RX1 and immune checkpoint molecules in metastatic PDAC samples. Bars represent mean ± standard deviation in ( d ). * P < 0.05, ** P < 0.01, and *** P < 0.001, by one-way ANOVA and Tukey’s multiple comparisons test ( d left), or Student’s t test ( d right). Source data are provided as a Source data file.

    Journal: Nature Communications

    Article Title: Identification of a subset of immunosuppressive P2RX1-negative neutrophils in pancreatic cancer liver metastasis

    doi: 10.1038/s41467-020-20447-y

    Figure Lengend Snippet: a Volcano plots of differential gene expression in 145 primary PDAC, 46 adjacent pancreases, 25 liver metastases and 27 adjacent livers. Red dots represent upregulated immune-related genes, and blue dots represent downregulated immune-related genes. b Immunome analyses of 26 infiltrating immune cell types in adjacent pancreas, primary PDAC, adjacent liver tissue and metastatic PDAC samples. c GO Biological Process analyses of differentially expressed genes between adjacent liver tissue and metastatic PDAC samples. d Expression analyses of P2RX1 in the adjacent pancreas, primary PDAC, adjacent liver tissue and metastatic PDAC samples from the GSE71729 and Renji cohorts. e Correlation analyses between P2RX1 and immune checkpoint molecules in metastatic PDAC samples. Bars represent mean ± standard deviation in ( d ). * P < 0.05, ** P < 0.01, and *** P < 0.001, by one-way ANOVA and Tukey’s multiple comparisons test ( d left), or Student’s t test ( d right). Source data are provided as a Source data file.

    Article Snippet: For immunofluorescence staining, paraffin sections were dewaxed with gradient ethanol, steam heated for antigen retrieval in citrate-based buffer, blocked with PBS containing 10%BSA for 1 h, stained with anti-P2RX1 (Alomone, 1:200), and CD66b (Biologend, 1:1000), Ly6G (Biolegend, 1:1000), or citrullinated histone H3 (CitH3) (Alomone, 1:200) overnight and secondary antibody (Abcam, 1:1000) for 2 hr and finally mounted in DAPI-containing media (Vector Labs) for imaging on a laser scanning confocal microscope (Leica SP8 LAS X).

    Techniques: Expressing, Standard Deviation

    a , b P2rx1 −/− mice were generated using CRISPR/Cas9 system. Schematic diagram was shown in ( a ) and genotyping results were shown in ( b ) (representative result from three independent experiments). c , d KPC cells were intrasplenically injected to seed livers of WT and P2rx1 −/− mice, and in vivo imaging was performed at sequential times. Representative images are shown in ( c ), and quantitative results are shown in ( d ) ( n = 6 per group, three independent experiments). e Representative images of liver metastatic samples harvested at day 17. f Liver weight of liver metastatic samples was measured at day 17 ( n = 5 per group, two independent experiments). g Survival analysis of liver metastatic WT or P2rx1 −/− mice within a duration of 5 weeks ( n = 10 per group, two independent experiments). h Normal liver (D0) and two sequential stages (D3 and D17) of liver metastases in WT and P2rx1 −/− mice were harvested for RNA-seq, and PCA analyses were performed ( n = 3 for D0, n = 4 for D3 and D17). i Representative Ki67 immunohistochemical staining of WT and P2rx1 −/− liver metastases at day 17 ( n = 4 per group, two independent experiments). 100 μm of scale bar for low power fields, 25 μm of scale bar for high power fields. j Heatmap of immune checkpoint molecules in liver metastases of WT or P2rx1 −/− mice at D3 and D17 ( n = 4 per group). Bars represent mean ± standard deviation in ( d , f ). P values are derived from two-sided Student’s t test ( d , f ), or log-rank test ( g ). Source data are provided as a Source data file.

    Journal: Nature Communications

    Article Title: Identification of a subset of immunosuppressive P2RX1-negative neutrophils in pancreatic cancer liver metastasis

    doi: 10.1038/s41467-020-20447-y

    Figure Lengend Snippet: a , b P2rx1 −/− mice were generated using CRISPR/Cas9 system. Schematic diagram was shown in ( a ) and genotyping results were shown in ( b ) (representative result from three independent experiments). c , d KPC cells were intrasplenically injected to seed livers of WT and P2rx1 −/− mice, and in vivo imaging was performed at sequential times. Representative images are shown in ( c ), and quantitative results are shown in ( d ) ( n = 6 per group, three independent experiments). e Representative images of liver metastatic samples harvested at day 17. f Liver weight of liver metastatic samples was measured at day 17 ( n = 5 per group, two independent experiments). g Survival analysis of liver metastatic WT or P2rx1 −/− mice within a duration of 5 weeks ( n = 10 per group, two independent experiments). h Normal liver (D0) and two sequential stages (D3 and D17) of liver metastases in WT and P2rx1 −/− mice were harvested for RNA-seq, and PCA analyses were performed ( n = 3 for D0, n = 4 for D3 and D17). i Representative Ki67 immunohistochemical staining of WT and P2rx1 −/− liver metastases at day 17 ( n = 4 per group, two independent experiments). 100 μm of scale bar for low power fields, 25 μm of scale bar for high power fields. j Heatmap of immune checkpoint molecules in liver metastases of WT or P2rx1 −/− mice at D3 and D17 ( n = 4 per group). Bars represent mean ± standard deviation in ( d , f ). P values are derived from two-sided Student’s t test ( d , f ), or log-rank test ( g ). Source data are provided as a Source data file.

    Article Snippet: For immunofluorescence staining, paraffin sections were dewaxed with gradient ethanol, steam heated for antigen retrieval in citrate-based buffer, blocked with PBS containing 10%BSA for 1 h, stained with anti-P2RX1 (Alomone, 1:200), and CD66b (Biologend, 1:1000), Ly6G (Biolegend, 1:1000), or citrullinated histone H3 (CitH3) (Alomone, 1:200) overnight and secondary antibody (Abcam, 1:1000) for 2 hr and finally mounted in DAPI-containing media (Vector Labs) for imaging on a laser scanning confocal microscope (Leica SP8 LAS X).

    Techniques: Generated, CRISPR, Injection, In Vivo Imaging, RNA Sequencing Assay, Immunohistochemical staining, Staining, Standard Deviation, Derivative Assay

    a Single cell suspension was obtained from mouse spleen and expression of P2RX1 was determined in indicated immune cell types ( n = 3 per group, two independent experiments). Left dotted line indicates the mean of negative control (NC) (secondary antibody only), and right dotted line indicates the mean of Ly6G+ cells. b KPC cells were intrasplenically injected to seed livers of WT mice. A single cell suspension was obtained from liver metastases and adjacent liver tissues of WT mice at day 17. The frequency of CD45+P2RX1+ cells was determined by flow cytometry ( n = 4 per group, three independent experiments). c KPC cells were intrasplenically injected to seed livers of WT mice. Immune cells were enriched from single cell suspension of liver metastases and adjacent liver tissues at day 17. P2RX1 expression in the indicated immune cell types was determined by flow cytometry ( n = 4 per group, three independent experiments). d Representative images of H&E staining and immunofluorescence staining of P2RX1 (Green), Ly6G (Red) and DAPI (Blue) in KPC mice spontaneous liver metastases (representative results from six independent experiments). 50 μm scale bar for low power fields and 20 μm scale bar for high power fields. e , f H&E staining and immunofluorescence staining of P2RX1 (green), CD66b (red) and DAPI (blue) in a total of 20 clinical PDAC liver metastasis samples were performed. Representative images are shown in ( e ), and the percentages of P2RX1-CD66b+ cells are shown in ( f ). 50 μm scale bar for low power fields and 20 μm scale bar for high power fields. g , h KPC cells were intrasplenically injected to seed livers of BM chimeras: WT → WT, P2rx1 −/− → P2rx1 −/− , WT → P2rx1 −/− , and P2rx1 −/− → WT. Neutrophils were depleted in WT → P2rx1 −/− and P2rx1 −/− → WT mice by intraperitoneal injection of anti-Ly6G (clone 1A8) antibody. At day 17, liver metastases were analyzed by in vivo imaging ( g , n = 5 for WT → WT, P2rx1 −/− → P2rx1 −/− and WT → P2rx1 −/− groups, n = 4 for WT → P2rx1 −/− + anti-Ly6G, P2rx1 −/− → WT and P2rx1 −/− → WT + anti-Ly6G groups, two independent experiments), and representative images of liver metastatic samples were shown ( h ). Bars represent mean ± standard deviation in ( f , g ). P values are derived from two-sided Student’s t test ( c , f ), or one-way ANOVA and Tukey’s multiple comparisons test ( g ). Source data are provided as a Source data file.

    Journal: Nature Communications

    Article Title: Identification of a subset of immunosuppressive P2RX1-negative neutrophils in pancreatic cancer liver metastasis

    doi: 10.1038/s41467-020-20447-y

    Figure Lengend Snippet: a Single cell suspension was obtained from mouse spleen and expression of P2RX1 was determined in indicated immune cell types ( n = 3 per group, two independent experiments). Left dotted line indicates the mean of negative control (NC) (secondary antibody only), and right dotted line indicates the mean of Ly6G+ cells. b KPC cells were intrasplenically injected to seed livers of WT mice. A single cell suspension was obtained from liver metastases and adjacent liver tissues of WT mice at day 17. The frequency of CD45+P2RX1+ cells was determined by flow cytometry ( n = 4 per group, three independent experiments). c KPC cells were intrasplenically injected to seed livers of WT mice. Immune cells were enriched from single cell suspension of liver metastases and adjacent liver tissues at day 17. P2RX1 expression in the indicated immune cell types was determined by flow cytometry ( n = 4 per group, three independent experiments). d Representative images of H&E staining and immunofluorescence staining of P2RX1 (Green), Ly6G (Red) and DAPI (Blue) in KPC mice spontaneous liver metastases (representative results from six independent experiments). 50 μm scale bar for low power fields and 20 μm scale bar for high power fields. e , f H&E staining and immunofluorescence staining of P2RX1 (green), CD66b (red) and DAPI (blue) in a total of 20 clinical PDAC liver metastasis samples were performed. Representative images are shown in ( e ), and the percentages of P2RX1-CD66b+ cells are shown in ( f ). 50 μm scale bar for low power fields and 20 μm scale bar for high power fields. g , h KPC cells were intrasplenically injected to seed livers of BM chimeras: WT → WT, P2rx1 −/− → P2rx1 −/− , WT → P2rx1 −/− , and P2rx1 −/− → WT. Neutrophils were depleted in WT → P2rx1 −/− and P2rx1 −/− → WT mice by intraperitoneal injection of anti-Ly6G (clone 1A8) antibody. At day 17, liver metastases were analyzed by in vivo imaging ( g , n = 5 for WT → WT, P2rx1 −/− → P2rx1 −/− and WT → P2rx1 −/− groups, n = 4 for WT → P2rx1 −/− + anti-Ly6G, P2rx1 −/− → WT and P2rx1 −/− → WT + anti-Ly6G groups, two independent experiments), and representative images of liver metastatic samples were shown ( h ). Bars represent mean ± standard deviation in ( f , g ). P values are derived from two-sided Student’s t test ( c , f ), or one-way ANOVA and Tukey’s multiple comparisons test ( g ). Source data are provided as a Source data file.

    Article Snippet: For immunofluorescence staining, paraffin sections were dewaxed with gradient ethanol, steam heated for antigen retrieval in citrate-based buffer, blocked with PBS containing 10%BSA for 1 h, stained with anti-P2RX1 (Alomone, 1:200), and CD66b (Biologend, 1:1000), Ly6G (Biolegend, 1:1000), or citrullinated histone H3 (CitH3) (Alomone, 1:200) overnight and secondary antibody (Abcam, 1:1000) for 2 hr and finally mounted in DAPI-containing media (Vector Labs) for imaging on a laser scanning confocal microscope (Leica SP8 LAS X).

    Techniques: Expressing, Negative Control, Injection, Flow Cytometry, Staining, Immunofluorescence, In Vivo Imaging, Standard Deviation, Derivative Assay

    a – g KPC cell was intrasplenically injected to seed livers of WT mice. Bone marrow (BM) and peripheral blood (PB) were obtained at day 17. Frequencies of neutrophils, P2RX1− neutrophils, and CXCR4+ neutrophils were determined by flow cytometry and quantitative results were shown ( n = 4 per group, three independent experiments). h Bone marrow neutrophils were isolated from WT mice and stimulated with indicated stimulus. RNA-seq were performed and Log 2 (FPKM+0.001) value of purinergic receptors were shown ( n = 1 per group). Bars represent mean ± standard deviation in ( e – g ). P values are derived from two-sided Student’s t test ( e – g ). Source data are provided as a Source data file.

    Journal: Nature Communications

    Article Title: Identification of a subset of immunosuppressive P2RX1-negative neutrophils in pancreatic cancer liver metastasis

    doi: 10.1038/s41467-020-20447-y

    Figure Lengend Snippet: a – g KPC cell was intrasplenically injected to seed livers of WT mice. Bone marrow (BM) and peripheral blood (PB) were obtained at day 17. Frequencies of neutrophils, P2RX1− neutrophils, and CXCR4+ neutrophils were determined by flow cytometry and quantitative results were shown ( n = 4 per group, three independent experiments). h Bone marrow neutrophils were isolated from WT mice and stimulated with indicated stimulus. RNA-seq were performed and Log 2 (FPKM+0.001) value of purinergic receptors were shown ( n = 1 per group). Bars represent mean ± standard deviation in ( e – g ). P values are derived from two-sided Student’s t test ( e – g ). Source data are provided as a Source data file.

    Article Snippet: For immunofluorescence staining, paraffin sections were dewaxed with gradient ethanol, steam heated for antigen retrieval in citrate-based buffer, blocked with PBS containing 10%BSA for 1 h, stained with anti-P2RX1 (Alomone, 1:200), and CD66b (Biologend, 1:1000), Ly6G (Biolegend, 1:1000), or citrullinated histone H3 (CitH3) (Alomone, 1:200) overnight and secondary antibody (Abcam, 1:1000) for 2 hr and finally mounted in DAPI-containing media (Vector Labs) for imaging on a laser scanning confocal microscope (Leica SP8 LAS X).

    Techniques: Injection, Flow Cytometry, Isolation, RNA Sequencing Assay, Standard Deviation, Derivative Assay

    a , b KPC cells were intrasplenically injected to seed livers of WT and P2rx1 −/− mice. A single cell suspension was obtained from liver metastases at day 17. Then, P2RX1+ neutrophils were purified from WT mice, and P2rx1 −/− neutrophils were purified from P2rx1 −/− mice for RNA sequencing. The results of KEGG analysis are shown in ( a ), and comparative expression of genes is shown in ( b ) ( n = 1 per group). c Bone marrow neutrophils were isolated from WT and P2rx1 −/− mice and stimulated with tumor conditioned medium (TCM). The ECAR and OCR were then measured by a Seahorse assay in ( c ), and PD-L1 and TNF-α were detected by RT-qPCR in ( d ) ( n = 4 per group, two independent experiments). Glc, glucose; O (ECAR), oligomycin; 2-DG, 2-deoxyglucose; O (OCR), oligomycin; F, FCCP (carbonyl cyanide 4-[trifluoromethoxy] phenylhydrazone); A & R, antimycin A and rotenone. Bars represent mean ± standard deviation in ( c , d ). P values are derived from two-sided Student’s t test ( d ). Source data are provided as a Source data file.

    Journal: Nature Communications

    Article Title: Identification of a subset of immunosuppressive P2RX1-negative neutrophils in pancreatic cancer liver metastasis

    doi: 10.1038/s41467-020-20447-y

    Figure Lengend Snippet: a , b KPC cells were intrasplenically injected to seed livers of WT and P2rx1 −/− mice. A single cell suspension was obtained from liver metastases at day 17. Then, P2RX1+ neutrophils were purified from WT mice, and P2rx1 −/− neutrophils were purified from P2rx1 −/− mice for RNA sequencing. The results of KEGG analysis are shown in ( a ), and comparative expression of genes is shown in ( b ) ( n = 1 per group). c Bone marrow neutrophils were isolated from WT and P2rx1 −/− mice and stimulated with tumor conditioned medium (TCM). The ECAR and OCR were then measured by a Seahorse assay in ( c ), and PD-L1 and TNF-α were detected by RT-qPCR in ( d ) ( n = 4 per group, two independent experiments). Glc, glucose; O (ECAR), oligomycin; 2-DG, 2-deoxyglucose; O (OCR), oligomycin; F, FCCP (carbonyl cyanide 4-[trifluoromethoxy] phenylhydrazone); A & R, antimycin A and rotenone. Bars represent mean ± standard deviation in ( c , d ). P values are derived from two-sided Student’s t test ( d ). Source data are provided as a Source data file.

    Article Snippet: For immunofluorescence staining, paraffin sections were dewaxed with gradient ethanol, steam heated for antigen retrieval in citrate-based buffer, blocked with PBS containing 10%BSA for 1 h, stained with anti-P2RX1 (Alomone, 1:200), and CD66b (Biologend, 1:1000), Ly6G (Biolegend, 1:1000), or citrullinated histone H3 (CitH3) (Alomone, 1:200) overnight and secondary antibody (Abcam, 1:1000) for 2 hr and finally mounted in DAPI-containing media (Vector Labs) for imaging on a laser scanning confocal microscope (Leica SP8 LAS X).

    Techniques: Injection, Purification, RNA Sequencing Assay, Expressing, Isolation, Quantitative RT-PCR, Standard Deviation, Derivative Assay

    a Gene set enrichment analysis comparing RNA-seq data of P2RX1+ neutrophils and P2rx1 −/− neutrophils based on Nrf2 target genes. The p value and normalized enrichment score (NES) were shown. b , c Single-cell suspensions were obtained from liver metastases of WT and P2rx1 −/− mice at day 17. Intracellular Nrf2 was stained and detected by flow cytometry ( b ) or laser scanning confocal microscopy ( c ) in WT P2RX1+ neutrophils and P2rx1 −/− neutrophils ( n = 4 per group, three independent experiments). The scale bar is 2.5 μm. d Bone marrow neutrophils were isolated from WT and P2rx1 −/− mice and stimulated with LPS + IFN-γ in the presence or absence of a Nrf2 inhibitor. The ECAR was then measured by a Seahorse assay ( n = 4 per group, two independent experiments). Glc, glucose; O, oligomycin; 2-DG, 2-deoxyglucose. e Bone marrow neutrophils were isolated from WT and P2rx1 −/− mice and stimulated with IL-4 in the presence or absence of a Nrf2 inhibitor. The OCR was then measured by a Seahorse assay ( n = 4 per group, two independent experiments). O, oligomycin; F, FCCP (carbonyl cyanide 4-[trifluoromethoxy] phenylhydrazone); A & R, antimycin A and rotenone. f Bone marrow neutrophils were isolated from WT or P2rx1 −/− mice and stimulated with LPS + IFN-γ and an inhibitor Nrf2 inhibitor. IL-1β and TNF-α were determined by RT-qPCR ( n = 4 per group, three independent experiments). Bars represent mean ± standard deviation in ( d – f ). P values are derived from permutation test ( a ), two-sided Student’s t test ( f ). Source data are provided as a Source data file.

    Journal: Nature Communications

    Article Title: Identification of a subset of immunosuppressive P2RX1-negative neutrophils in pancreatic cancer liver metastasis

    doi: 10.1038/s41467-020-20447-y

    Figure Lengend Snippet: a Gene set enrichment analysis comparing RNA-seq data of P2RX1+ neutrophils and P2rx1 −/− neutrophils based on Nrf2 target genes. The p value and normalized enrichment score (NES) were shown. b , c Single-cell suspensions were obtained from liver metastases of WT and P2rx1 −/− mice at day 17. Intracellular Nrf2 was stained and detected by flow cytometry ( b ) or laser scanning confocal microscopy ( c ) in WT P2RX1+ neutrophils and P2rx1 −/− neutrophils ( n = 4 per group, three independent experiments). The scale bar is 2.5 μm. d Bone marrow neutrophils were isolated from WT and P2rx1 −/− mice and stimulated with LPS + IFN-γ in the presence or absence of a Nrf2 inhibitor. The ECAR was then measured by a Seahorse assay ( n = 4 per group, two independent experiments). Glc, glucose; O, oligomycin; 2-DG, 2-deoxyglucose. e Bone marrow neutrophils were isolated from WT and P2rx1 −/− mice and stimulated with IL-4 in the presence or absence of a Nrf2 inhibitor. The OCR was then measured by a Seahorse assay ( n = 4 per group, two independent experiments). O, oligomycin; F, FCCP (carbonyl cyanide 4-[trifluoromethoxy] phenylhydrazone); A & R, antimycin A and rotenone. f Bone marrow neutrophils were isolated from WT or P2rx1 −/− mice and stimulated with LPS + IFN-γ and an inhibitor Nrf2 inhibitor. IL-1β and TNF-α were determined by RT-qPCR ( n = 4 per group, three independent experiments). Bars represent mean ± standard deviation in ( d – f ). P values are derived from permutation test ( a ), two-sided Student’s t test ( f ). Source data are provided as a Source data file.

    Article Snippet: For immunofluorescence staining, paraffin sections were dewaxed with gradient ethanol, steam heated for antigen retrieval in citrate-based buffer, blocked with PBS containing 10%BSA for 1 h, stained with anti-P2RX1 (Alomone, 1:200), and CD66b (Biologend, 1:1000), Ly6G (Biolegend, 1:1000), or citrullinated histone H3 (CitH3) (Alomone, 1:200) overnight and secondary antibody (Abcam, 1:1000) for 2 hr and finally mounted in DAPI-containing media (Vector Labs) for imaging on a laser scanning confocal microscope (Leica SP8 LAS X).

    Techniques: RNA Sequencing Assay, Staining, Flow Cytometry, Confocal Microscopy, Isolation, Quantitative RT-PCR, Standard Deviation, Derivative Assay

    a A single cell suspension was obtained from liver metastases of WT and P2rx1 −/− mice at day 17. Flow cytometry was performed to detect the frequency of CD8+PD-1+ (upper) and Ly6G+PD-L1 + or Ly6G+PD-L1− (lower) cells. P2RX1 expression was further determined in Ly6G+PD-L1+ and Ly6G+PD-L1− cells from WT mice ( n = 4 per group, three independent experiments). b Bone marrow neutrophils were isolated from WT and P2rx1 −/− mice and stimulated with tumor conditioned medium (TCM) or GM-CSF in the presence of a Nrf2 inhibitor or anti-GM-CSF neutralizing antibody. PD-L1 expression was detected by flow cytometry ( n = 4 per group, three independent experiments). c Integrative Genomics Viewer (IGV) was used to predict the two peaks that PD-L1 gene might be mediated by Nrf2 (upper). Bone marrow neutrophils were isolated from WT and P2rx1 −/− mice and stimulated with GM-CSF. Binding of PD-L1 gene by Nrf2 was detected by ChIP-PCR ( n = 3 per group, two independent experiments). d Bone marrow neutrophils were isolated from WT and P2rx1 −/− mice and stimulated with GM-CSF. Intracellular ROS was detected by flow cytometry ( n = 4 per group, three independent experiments). e Antigen activated CTLs was co-cultured with GM-CSF primed WT or P2rx1 −/− neutrophils. Cell proliferation was analyzed with CSFE staining in the presence or absence of anti-PD-1 neutralizing antibody ( n = 4 per group, three independent experiments). f KPC cells were transfected with empty lentiviral vector (KPC-LV) or OVA (KPC-OVA). Antigen-activated CTLs were co-cultured with KPC-LV or KPC-OVA cells in the presence of GM-CSF-primed WT or P2rx1 −/− neutrophils. Cytotoxicity was determined in the presence or absence of anti-PD-1 neutralizing antibody by counting the number of PI+ cells ( n = 4 per group, three independent experiments). Bars represent mean ± standard deviation in ( f ). P values are derived from one-way ANOVA and Tukey’s multiple comparisons test ( f ). Source data are provided as a Source data file.

    Journal: Nature Communications

    Article Title: Identification of a subset of immunosuppressive P2RX1-negative neutrophils in pancreatic cancer liver metastasis

    doi: 10.1038/s41467-020-20447-y

    Figure Lengend Snippet: a A single cell suspension was obtained from liver metastases of WT and P2rx1 −/− mice at day 17. Flow cytometry was performed to detect the frequency of CD8+PD-1+ (upper) and Ly6G+PD-L1 + or Ly6G+PD-L1− (lower) cells. P2RX1 expression was further determined in Ly6G+PD-L1+ and Ly6G+PD-L1− cells from WT mice ( n = 4 per group, three independent experiments). b Bone marrow neutrophils were isolated from WT and P2rx1 −/− mice and stimulated with tumor conditioned medium (TCM) or GM-CSF in the presence of a Nrf2 inhibitor or anti-GM-CSF neutralizing antibody. PD-L1 expression was detected by flow cytometry ( n = 4 per group, three independent experiments). c Integrative Genomics Viewer (IGV) was used to predict the two peaks that PD-L1 gene might be mediated by Nrf2 (upper). Bone marrow neutrophils were isolated from WT and P2rx1 −/− mice and stimulated with GM-CSF. Binding of PD-L1 gene by Nrf2 was detected by ChIP-PCR ( n = 3 per group, two independent experiments). d Bone marrow neutrophils were isolated from WT and P2rx1 −/− mice and stimulated with GM-CSF. Intracellular ROS was detected by flow cytometry ( n = 4 per group, three independent experiments). e Antigen activated CTLs was co-cultured with GM-CSF primed WT or P2rx1 −/− neutrophils. Cell proliferation was analyzed with CSFE staining in the presence or absence of anti-PD-1 neutralizing antibody ( n = 4 per group, three independent experiments). f KPC cells were transfected with empty lentiviral vector (KPC-LV) or OVA (KPC-OVA). Antigen-activated CTLs were co-cultured with KPC-LV or KPC-OVA cells in the presence of GM-CSF-primed WT or P2rx1 −/− neutrophils. Cytotoxicity was determined in the presence or absence of anti-PD-1 neutralizing antibody by counting the number of PI+ cells ( n = 4 per group, three independent experiments). Bars represent mean ± standard deviation in ( f ). P values are derived from one-way ANOVA and Tukey’s multiple comparisons test ( f ). Source data are provided as a Source data file.

    Article Snippet: For immunofluorescence staining, paraffin sections were dewaxed with gradient ethanol, steam heated for antigen retrieval in citrate-based buffer, blocked with PBS containing 10%BSA for 1 h, stained with anti-P2RX1 (Alomone, 1:200), and CD66b (Biologend, 1:1000), Ly6G (Biolegend, 1:1000), or citrullinated histone H3 (CitH3) (Alomone, 1:200) overnight and secondary antibody (Abcam, 1:1000) for 2 hr and finally mounted in DAPI-containing media (Vector Labs) for imaging on a laser scanning confocal microscope (Leica SP8 LAS X).

    Techniques: Flow Cytometry, Expressing, Isolation, Binding Assay, Cell Culture, Staining, Transfection, Plasmid Preparation, Standard Deviation, Derivative Assay

    a Volcano plots of differential gene expression in 145 primary PDAC, 46 adjacent pancreases, 25 liver metastases and 27 adjacent livers. Red dots represent upregulated immune-related genes, and blue dots represent downregulated immune-related genes. b Immunome analyses of 26 infiltrating immune cell types in adjacent pancreas, primary PDAC, adjacent liver tissue and metastatic PDAC samples. c GO Biological Process analyses of differentially expressed genes between adjacent liver tissue and metastatic PDAC samples. d Expression analyses of P2RX1 in the adjacent pancreas, primary PDAC, adjacent liver tissue and metastatic PDAC samples from the GSE71729 and Renji cohorts. e Correlation analyses between P2RX1 and immune checkpoint molecules in metastatic PDAC samples. Bars represent mean ± standard deviation in ( d ). * P < 0.05, ** P < 0.01, and *** P < 0.001, by one-way ANOVA and Tukey’s multiple comparisons test ( d left), or Student’s t test ( d right). Source data are provided as a Source data file.

    Journal: Nature Communications

    Article Title: Identification of a subset of immunosuppressive P2RX1-negative neutrophils in pancreatic cancer liver metastasis

    doi: 10.1038/s41467-020-20447-y

    Figure Lengend Snippet: a Volcano plots of differential gene expression in 145 primary PDAC, 46 adjacent pancreases, 25 liver metastases and 27 adjacent livers. Red dots represent upregulated immune-related genes, and blue dots represent downregulated immune-related genes. b Immunome analyses of 26 infiltrating immune cell types in adjacent pancreas, primary PDAC, adjacent liver tissue and metastatic PDAC samples. c GO Biological Process analyses of differentially expressed genes between adjacent liver tissue and metastatic PDAC samples. d Expression analyses of P2RX1 in the adjacent pancreas, primary PDAC, adjacent liver tissue and metastatic PDAC samples from the GSE71729 and Renji cohorts. e Correlation analyses between P2RX1 and immune checkpoint molecules in metastatic PDAC samples. Bars represent mean ± standard deviation in ( d ). * P < 0.05, ** P < 0.01, and *** P < 0.001, by one-way ANOVA and Tukey’s multiple comparisons test ( d left), or Student’s t test ( d right). Source data are provided as a Source data file.

    Article Snippet: For P2RX1 labeling (Alomone, 1:80), primary antibody was labeled for 30 min. After washing, fluorophore-conjugated secondary antibodies were labeled for another 30 min. Before sorting, SYTOX Blue (Thermo Fisher) was added to exclude dead cells.

    Techniques: Expressing, Standard Deviation

    a , b P2rx1 −/− mice were generated using CRISPR/Cas9 system. Schematic diagram was shown in ( a ) and genotyping results were shown in ( b ) (representative result from three independent experiments). c , d KPC cells were intrasplenically injected to seed livers of WT and P2rx1 −/− mice, and in vivo imaging was performed at sequential times. Representative images are shown in ( c ), and quantitative results are shown in ( d ) ( n = 6 per group, three independent experiments). e Representative images of liver metastatic samples harvested at day 17. f Liver weight of liver metastatic samples was measured at day 17 ( n = 5 per group, two independent experiments). g Survival analysis of liver metastatic WT or P2rx1 −/− mice within a duration of 5 weeks ( n = 10 per group, two independent experiments). h Normal liver (D0) and two sequential stages (D3 and D17) of liver metastases in WT and P2rx1 −/− mice were harvested for RNA-seq, and PCA analyses were performed ( n = 3 for D0, n = 4 for D3 and D17). i Representative Ki67 immunohistochemical staining of WT and P2rx1 −/− liver metastases at day 17 ( n = 4 per group, two independent experiments). 100 μm of scale bar for low power fields, 25 μm of scale bar for high power fields. j Heatmap of immune checkpoint molecules in liver metastases of WT or P2rx1 −/− mice at D3 and D17 ( n = 4 per group). Bars represent mean ± standard deviation in ( d , f ). P values are derived from two-sided Student’s t test ( d , f ), or log-rank test ( g ). Source data are provided as a Source data file.

    Journal: Nature Communications

    Article Title: Identification of a subset of immunosuppressive P2RX1-negative neutrophils in pancreatic cancer liver metastasis

    doi: 10.1038/s41467-020-20447-y

    Figure Lengend Snippet: a , b P2rx1 −/− mice were generated using CRISPR/Cas9 system. Schematic diagram was shown in ( a ) and genotyping results were shown in ( b ) (representative result from three independent experiments). c , d KPC cells were intrasplenically injected to seed livers of WT and P2rx1 −/− mice, and in vivo imaging was performed at sequential times. Representative images are shown in ( c ), and quantitative results are shown in ( d ) ( n = 6 per group, three independent experiments). e Representative images of liver metastatic samples harvested at day 17. f Liver weight of liver metastatic samples was measured at day 17 ( n = 5 per group, two independent experiments). g Survival analysis of liver metastatic WT or P2rx1 −/− mice within a duration of 5 weeks ( n = 10 per group, two independent experiments). h Normal liver (D0) and two sequential stages (D3 and D17) of liver metastases in WT and P2rx1 −/− mice were harvested for RNA-seq, and PCA analyses were performed ( n = 3 for D0, n = 4 for D3 and D17). i Representative Ki67 immunohistochemical staining of WT and P2rx1 −/− liver metastases at day 17 ( n = 4 per group, two independent experiments). 100 μm of scale bar for low power fields, 25 μm of scale bar for high power fields. j Heatmap of immune checkpoint molecules in liver metastases of WT or P2rx1 −/− mice at D3 and D17 ( n = 4 per group). Bars represent mean ± standard deviation in ( d , f ). P values are derived from two-sided Student’s t test ( d , f ), or log-rank test ( g ). Source data are provided as a Source data file.

    Article Snippet: For P2RX1 labeling (Alomone, 1:80), primary antibody was labeled for 30 min. After washing, fluorophore-conjugated secondary antibodies were labeled for another 30 min. Before sorting, SYTOX Blue (Thermo Fisher) was added to exclude dead cells.

    Techniques: Generated, CRISPR, Injection, In Vivo Imaging, RNA Sequencing Assay, Immunohistochemical staining, Staining, Standard Deviation, Derivative Assay

    a Single cell suspension was obtained from mouse spleen and expression of P2RX1 was determined in indicated immune cell types ( n = 3 per group, two independent experiments). Left dotted line indicates the mean of negative control (NC) (secondary antibody only), and right dotted line indicates the mean of Ly6G+ cells. b KPC cells were intrasplenically injected to seed livers of WT mice. A single cell suspension was obtained from liver metastases and adjacent liver tissues of WT mice at day 17. The frequency of CD45+P2RX1+ cells was determined by flow cytometry ( n = 4 per group, three independent experiments). c KPC cells were intrasplenically injected to seed livers of WT mice. Immune cells were enriched from single cell suspension of liver metastases and adjacent liver tissues at day 17. P2RX1 expression in the indicated immune cell types was determined by flow cytometry ( n = 4 per group, three independent experiments). d Representative images of H&E staining and immunofluorescence staining of P2RX1 (Green), Ly6G (Red) and DAPI (Blue) in KPC mice spontaneous liver metastases (representative results from six independent experiments). 50 μm scale bar for low power fields and 20 μm scale bar for high power fields. e , f H&E staining and immunofluorescence staining of P2RX1 (green), CD66b (red) and DAPI (blue) in a total of 20 clinical PDAC liver metastasis samples were performed. Representative images are shown in ( e ), and the percentages of P2RX1-CD66b+ cells are shown in ( f ). 50 μm scale bar for low power fields and 20 μm scale bar for high power fields. g , h KPC cells were intrasplenically injected to seed livers of BM chimeras: WT → WT, P2rx1 −/− → P2rx1 −/− , WT → P2rx1 −/− , and P2rx1 −/− → WT. Neutrophils were depleted in WT → P2rx1 −/− and P2rx1 −/− → WT mice by intraperitoneal injection of anti-Ly6G (clone 1A8) antibody. At day 17, liver metastases were analyzed by in vivo imaging ( g , n = 5 for WT → WT, P2rx1 −/− → P2rx1 −/− and WT → P2rx1 −/− groups, n = 4 for WT → P2rx1 −/− + anti-Ly6G, P2rx1 −/− → WT and P2rx1 −/− → WT + anti-Ly6G groups, two independent experiments), and representative images of liver metastatic samples were shown ( h ). Bars represent mean ± standard deviation in ( f , g ). P values are derived from two-sided Student’s t test ( c , f ), or one-way ANOVA and Tukey’s multiple comparisons test ( g ). Source data are provided as a Source data file.

    Journal: Nature Communications

    Article Title: Identification of a subset of immunosuppressive P2RX1-negative neutrophils in pancreatic cancer liver metastasis

    doi: 10.1038/s41467-020-20447-y

    Figure Lengend Snippet: a Single cell suspension was obtained from mouse spleen and expression of P2RX1 was determined in indicated immune cell types ( n = 3 per group, two independent experiments). Left dotted line indicates the mean of negative control (NC) (secondary antibody only), and right dotted line indicates the mean of Ly6G+ cells. b KPC cells were intrasplenically injected to seed livers of WT mice. A single cell suspension was obtained from liver metastases and adjacent liver tissues of WT mice at day 17. The frequency of CD45+P2RX1+ cells was determined by flow cytometry ( n = 4 per group, three independent experiments). c KPC cells were intrasplenically injected to seed livers of WT mice. Immune cells were enriched from single cell suspension of liver metastases and adjacent liver tissues at day 17. P2RX1 expression in the indicated immune cell types was determined by flow cytometry ( n = 4 per group, three independent experiments). d Representative images of H&E staining and immunofluorescence staining of P2RX1 (Green), Ly6G (Red) and DAPI (Blue) in KPC mice spontaneous liver metastases (representative results from six independent experiments). 50 μm scale bar for low power fields and 20 μm scale bar for high power fields. e , f H&E staining and immunofluorescence staining of P2RX1 (green), CD66b (red) and DAPI (blue) in a total of 20 clinical PDAC liver metastasis samples were performed. Representative images are shown in ( e ), and the percentages of P2RX1-CD66b+ cells are shown in ( f ). 50 μm scale bar for low power fields and 20 μm scale bar for high power fields. g , h KPC cells were intrasplenically injected to seed livers of BM chimeras: WT → WT, P2rx1 −/− → P2rx1 −/− , WT → P2rx1 −/− , and P2rx1 −/− → WT. Neutrophils were depleted in WT → P2rx1 −/− and P2rx1 −/− → WT mice by intraperitoneal injection of anti-Ly6G (clone 1A8) antibody. At day 17, liver metastases were analyzed by in vivo imaging ( g , n = 5 for WT → WT, P2rx1 −/− → P2rx1 −/− and WT → P2rx1 −/− groups, n = 4 for WT → P2rx1 −/− + anti-Ly6G, P2rx1 −/− → WT and P2rx1 −/− → WT + anti-Ly6G groups, two independent experiments), and representative images of liver metastatic samples were shown ( h ). Bars represent mean ± standard deviation in ( f , g ). P values are derived from two-sided Student’s t test ( c , f ), or one-way ANOVA and Tukey’s multiple comparisons test ( g ). Source data are provided as a Source data file.

    Article Snippet: For P2RX1 labeling (Alomone, 1:80), primary antibody was labeled for 30 min. After washing, fluorophore-conjugated secondary antibodies were labeled for another 30 min. Before sorting, SYTOX Blue (Thermo Fisher) was added to exclude dead cells.

    Techniques: Expressing, Negative Control, Injection, Flow Cytometry, Staining, Immunofluorescence, In Vivo Imaging, Standard Deviation, Derivative Assay

    a – g KPC cell was intrasplenically injected to seed livers of WT mice. Bone marrow (BM) and peripheral blood (PB) were obtained at day 17. Frequencies of neutrophils, P2RX1− neutrophils, and CXCR4+ neutrophils were determined by flow cytometry and quantitative results were shown ( n = 4 per group, three independent experiments). h Bone marrow neutrophils were isolated from WT mice and stimulated with indicated stimulus. RNA-seq were performed and Log 2 (FPKM+0.001) value of purinergic receptors were shown ( n = 1 per group). Bars represent mean ± standard deviation in ( e – g ). P values are derived from two-sided Student’s t test ( e – g ). Source data are provided as a Source data file.

    Journal: Nature Communications

    Article Title: Identification of a subset of immunosuppressive P2RX1-negative neutrophils in pancreatic cancer liver metastasis

    doi: 10.1038/s41467-020-20447-y

    Figure Lengend Snippet: a – g KPC cell was intrasplenically injected to seed livers of WT mice. Bone marrow (BM) and peripheral blood (PB) were obtained at day 17. Frequencies of neutrophils, P2RX1− neutrophils, and CXCR4+ neutrophils were determined by flow cytometry and quantitative results were shown ( n = 4 per group, three independent experiments). h Bone marrow neutrophils were isolated from WT mice and stimulated with indicated stimulus. RNA-seq were performed and Log 2 (FPKM+0.001) value of purinergic receptors were shown ( n = 1 per group). Bars represent mean ± standard deviation in ( e – g ). P values are derived from two-sided Student’s t test ( e – g ). Source data are provided as a Source data file.

    Article Snippet: For P2RX1 labeling (Alomone, 1:80), primary antibody was labeled for 30 min. After washing, fluorophore-conjugated secondary antibodies were labeled for another 30 min. Before sorting, SYTOX Blue (Thermo Fisher) was added to exclude dead cells.

    Techniques: Injection, Flow Cytometry, Isolation, RNA Sequencing Assay, Standard Deviation, Derivative Assay

    a , b KPC cells were intrasplenically injected to seed livers of WT and P2rx1 −/− mice. A single cell suspension was obtained from liver metastases at day 17. Then, P2RX1+ neutrophils were purified from WT mice, and P2rx1 −/− neutrophils were purified from P2rx1 −/− mice for RNA sequencing. The results of KEGG analysis are shown in ( a ), and comparative expression of genes is shown in ( b ) ( n = 1 per group). c Bone marrow neutrophils were isolated from WT and P2rx1 −/− mice and stimulated with tumor conditioned medium (TCM). The ECAR and OCR were then measured by a Seahorse assay in ( c ), and PD-L1 and TNF-α were detected by RT-qPCR in ( d ) ( n = 4 per group, two independent experiments). Glc, glucose; O (ECAR), oligomycin; 2-DG, 2-deoxyglucose; O (OCR), oligomycin; F, FCCP (carbonyl cyanide 4-[trifluoromethoxy] phenylhydrazone); A & R, antimycin A and rotenone. Bars represent mean ± standard deviation in ( c , d ). P values are derived from two-sided Student’s t test ( d ). Source data are provided as a Source data file.

    Journal: Nature Communications

    Article Title: Identification of a subset of immunosuppressive P2RX1-negative neutrophils in pancreatic cancer liver metastasis

    doi: 10.1038/s41467-020-20447-y

    Figure Lengend Snippet: a , b KPC cells were intrasplenically injected to seed livers of WT and P2rx1 −/− mice. A single cell suspension was obtained from liver metastases at day 17. Then, P2RX1+ neutrophils were purified from WT mice, and P2rx1 −/− neutrophils were purified from P2rx1 −/− mice for RNA sequencing. The results of KEGG analysis are shown in ( a ), and comparative expression of genes is shown in ( b ) ( n = 1 per group). c Bone marrow neutrophils were isolated from WT and P2rx1 −/− mice and stimulated with tumor conditioned medium (TCM). The ECAR and OCR were then measured by a Seahorse assay in ( c ), and PD-L1 and TNF-α were detected by RT-qPCR in ( d ) ( n = 4 per group, two independent experiments). Glc, glucose; O (ECAR), oligomycin; 2-DG, 2-deoxyglucose; O (OCR), oligomycin; F, FCCP (carbonyl cyanide 4-[trifluoromethoxy] phenylhydrazone); A & R, antimycin A and rotenone. Bars represent mean ± standard deviation in ( c , d ). P values are derived from two-sided Student’s t test ( d ). Source data are provided as a Source data file.

    Article Snippet: For P2RX1 labeling (Alomone, 1:80), primary antibody was labeled for 30 min. After washing, fluorophore-conjugated secondary antibodies were labeled for another 30 min. Before sorting, SYTOX Blue (Thermo Fisher) was added to exclude dead cells.

    Techniques: Injection, Purification, RNA Sequencing Assay, Expressing, Isolation, Quantitative RT-PCR, Standard Deviation, Derivative Assay

    a Gene set enrichment analysis comparing RNA-seq data of P2RX1+ neutrophils and P2rx1 −/− neutrophils based on Nrf2 target genes. The p value and normalized enrichment score (NES) were shown. b , c Single-cell suspensions were obtained from liver metastases of WT and P2rx1 −/− mice at day 17. Intracellular Nrf2 was stained and detected by flow cytometry ( b ) or laser scanning confocal microscopy ( c ) in WT P2RX1+ neutrophils and P2rx1 −/− neutrophils ( n = 4 per group, three independent experiments). The scale bar is 2.5 μm. d Bone marrow neutrophils were isolated from WT and P2rx1 −/− mice and stimulated with LPS + IFN-γ in the presence or absence of a Nrf2 inhibitor. The ECAR was then measured by a Seahorse assay ( n = 4 per group, two independent experiments). Glc, glucose; O, oligomycin; 2-DG, 2-deoxyglucose. e Bone marrow neutrophils were isolated from WT and P2rx1 −/− mice and stimulated with IL-4 in the presence or absence of a Nrf2 inhibitor. The OCR was then measured by a Seahorse assay ( n = 4 per group, two independent experiments). O, oligomycin; F, FCCP (carbonyl cyanide 4-[trifluoromethoxy] phenylhydrazone); A & R, antimycin A and rotenone. f Bone marrow neutrophils were isolated from WT or P2rx1 −/− mice and stimulated with LPS + IFN-γ and an inhibitor Nrf2 inhibitor. IL-1β and TNF-α were determined by RT-qPCR ( n = 4 per group, three independent experiments). Bars represent mean ± standard deviation in ( d – f ). P values are derived from permutation test ( a ), two-sided Student’s t test ( f ). Source data are provided as a Source data file.

    Journal: Nature Communications

    Article Title: Identification of a subset of immunosuppressive P2RX1-negative neutrophils in pancreatic cancer liver metastasis

    doi: 10.1038/s41467-020-20447-y

    Figure Lengend Snippet: a Gene set enrichment analysis comparing RNA-seq data of P2RX1+ neutrophils and P2rx1 −/− neutrophils based on Nrf2 target genes. The p value and normalized enrichment score (NES) were shown. b , c Single-cell suspensions were obtained from liver metastases of WT and P2rx1 −/− mice at day 17. Intracellular Nrf2 was stained and detected by flow cytometry ( b ) or laser scanning confocal microscopy ( c ) in WT P2RX1+ neutrophils and P2rx1 −/− neutrophils ( n = 4 per group, three independent experiments). The scale bar is 2.5 μm. d Bone marrow neutrophils were isolated from WT and P2rx1 −/− mice and stimulated with LPS + IFN-γ in the presence or absence of a Nrf2 inhibitor. The ECAR was then measured by a Seahorse assay ( n = 4 per group, two independent experiments). Glc, glucose; O, oligomycin; 2-DG, 2-deoxyglucose. e Bone marrow neutrophils were isolated from WT and P2rx1 −/− mice and stimulated with IL-4 in the presence or absence of a Nrf2 inhibitor. The OCR was then measured by a Seahorse assay ( n = 4 per group, two independent experiments). O, oligomycin; F, FCCP (carbonyl cyanide 4-[trifluoromethoxy] phenylhydrazone); A & R, antimycin A and rotenone. f Bone marrow neutrophils were isolated from WT or P2rx1 −/− mice and stimulated with LPS + IFN-γ and an inhibitor Nrf2 inhibitor. IL-1β and TNF-α were determined by RT-qPCR ( n = 4 per group, three independent experiments). Bars represent mean ± standard deviation in ( d – f ). P values are derived from permutation test ( a ), two-sided Student’s t test ( f ). Source data are provided as a Source data file.

    Article Snippet: For P2RX1 labeling (Alomone, 1:80), primary antibody was labeled for 30 min. After washing, fluorophore-conjugated secondary antibodies were labeled for another 30 min. Before sorting, SYTOX Blue (Thermo Fisher) was added to exclude dead cells.

    Techniques: RNA Sequencing Assay, Staining, Flow Cytometry, Confocal Microscopy, Isolation, Quantitative RT-PCR, Standard Deviation, Derivative Assay

    a A single cell suspension was obtained from liver metastases of WT and P2rx1 −/− mice at day 17. Flow cytometry was performed to detect the frequency of CD8+PD-1+ (upper) and Ly6G+PD-L1 + or Ly6G+PD-L1− (lower) cells. P2RX1 expression was further determined in Ly6G+PD-L1+ and Ly6G+PD-L1− cells from WT mice ( n = 4 per group, three independent experiments). b Bone marrow neutrophils were isolated from WT and P2rx1 −/− mice and stimulated with tumor conditioned medium (TCM) or GM-CSF in the presence of a Nrf2 inhibitor or anti-GM-CSF neutralizing antibody. PD-L1 expression was detected by flow cytometry ( n = 4 per group, three independent experiments). c Integrative Genomics Viewer (IGV) was used to predict the two peaks that PD-L1 gene might be mediated by Nrf2 (upper). Bone marrow neutrophils were isolated from WT and P2rx1 −/− mice and stimulated with GM-CSF. Binding of PD-L1 gene by Nrf2 was detected by ChIP-PCR ( n = 3 per group, two independent experiments). d Bone marrow neutrophils were isolated from WT and P2rx1 −/− mice and stimulated with GM-CSF. Intracellular ROS was detected by flow cytometry ( n = 4 per group, three independent experiments). e Antigen activated CTLs was co-cultured with GM-CSF primed WT or P2rx1 −/− neutrophils. Cell proliferation was analyzed with CSFE staining in the presence or absence of anti-PD-1 neutralizing antibody ( n = 4 per group, three independent experiments). f KPC cells were transfected with empty lentiviral vector (KPC-LV) or OVA (KPC-OVA). Antigen-activated CTLs were co-cultured with KPC-LV or KPC-OVA cells in the presence of GM-CSF-primed WT or P2rx1 −/− neutrophils. Cytotoxicity was determined in the presence or absence of anti-PD-1 neutralizing antibody by counting the number of PI+ cells ( n = 4 per group, three independent experiments). Bars represent mean ± standard deviation in ( f ). P values are derived from one-way ANOVA and Tukey’s multiple comparisons test ( f ). Source data are provided as a Source data file.

    Journal: Nature Communications

    Article Title: Identification of a subset of immunosuppressive P2RX1-negative neutrophils in pancreatic cancer liver metastasis

    doi: 10.1038/s41467-020-20447-y

    Figure Lengend Snippet: a A single cell suspension was obtained from liver metastases of WT and P2rx1 −/− mice at day 17. Flow cytometry was performed to detect the frequency of CD8+PD-1+ (upper) and Ly6G+PD-L1 + or Ly6G+PD-L1− (lower) cells. P2RX1 expression was further determined in Ly6G+PD-L1+ and Ly6G+PD-L1− cells from WT mice ( n = 4 per group, three independent experiments). b Bone marrow neutrophils were isolated from WT and P2rx1 −/− mice and stimulated with tumor conditioned medium (TCM) or GM-CSF in the presence of a Nrf2 inhibitor or anti-GM-CSF neutralizing antibody. PD-L1 expression was detected by flow cytometry ( n = 4 per group, three independent experiments). c Integrative Genomics Viewer (IGV) was used to predict the two peaks that PD-L1 gene might be mediated by Nrf2 (upper). Bone marrow neutrophils were isolated from WT and P2rx1 −/− mice and stimulated with GM-CSF. Binding of PD-L1 gene by Nrf2 was detected by ChIP-PCR ( n = 3 per group, two independent experiments). d Bone marrow neutrophils were isolated from WT and P2rx1 −/− mice and stimulated with GM-CSF. Intracellular ROS was detected by flow cytometry ( n = 4 per group, three independent experiments). e Antigen activated CTLs was co-cultured with GM-CSF primed WT or P2rx1 −/− neutrophils. Cell proliferation was analyzed with CSFE staining in the presence or absence of anti-PD-1 neutralizing antibody ( n = 4 per group, three independent experiments). f KPC cells were transfected with empty lentiviral vector (KPC-LV) or OVA (KPC-OVA). Antigen-activated CTLs were co-cultured with KPC-LV or KPC-OVA cells in the presence of GM-CSF-primed WT or P2rx1 −/− neutrophils. Cytotoxicity was determined in the presence or absence of anti-PD-1 neutralizing antibody by counting the number of PI+ cells ( n = 4 per group, three independent experiments). Bars represent mean ± standard deviation in ( f ). P values are derived from one-way ANOVA and Tukey’s multiple comparisons test ( f ). Source data are provided as a Source data file.

    Article Snippet: For P2RX1 labeling (Alomone, 1:80), primary antibody was labeled for 30 min. After washing, fluorophore-conjugated secondary antibodies were labeled for another 30 min. Before sorting, SYTOX Blue (Thermo Fisher) was added to exclude dead cells.

    Techniques: Flow Cytometry, Expressing, Isolation, Binding Assay, Cell Culture, Staining, Transfection, Plasmid Preparation, Standard Deviation, Derivative Assay

    Expression and function of purinergic receptors. The mean fluorescence intensities (MFI) of basal or 10 μM TRAP-6-stimulated receptor expression, detected by flow cytometry, are shown for P2Y1 ( A ), P2Y12 ( B ) and P2X1 ( C ). The function of the P2Y1 ( D ) and of the P2X1 ( E ) receptor was measured by calcium-induced fluorescence after selective stimulation, shown as mean values in relative fluorescence units (RFU). The function of the P2Y12 receptor ( F ) is expressed as platelet reactivity index (PRI) determined by the flow cytometric VASP assay. Mean ± SEM; n = 6; * p < 0.05 (for RT-APC in black, for C-APC in blue). C-APC, cold-stored apheresis-derived platelet concentrates; PB, peripheral blood; RT, room temperature; SEM, standard error of mean; TRAP-6, thrombin receptor activating peptide; VASP, vasodilator-stimulated phosphoprotein.

    Journal: TH Open: Companion Journal to Thrombosis and Haemostasis

    Article Title: The Impact of Cold Storage on Adenosine Diphosphate-Mediated Platelet Responsiveness

    doi: 10.1055/s-0040-1714254

    Figure Lengend Snippet: Expression and function of purinergic receptors. The mean fluorescence intensities (MFI) of basal or 10 μM TRAP-6-stimulated receptor expression, detected by flow cytometry, are shown for P2Y1 ( A ), P2Y12 ( B ) and P2X1 ( C ). The function of the P2Y1 ( D ) and of the P2X1 ( E ) receptor was measured by calcium-induced fluorescence after selective stimulation, shown as mean values in relative fluorescence units (RFU). The function of the P2Y12 receptor ( F ) is expressed as platelet reactivity index (PRI) determined by the flow cytometric VASP assay. Mean ± SEM; n = 6; * p < 0.05 (for RT-APC in black, for C-APC in blue). C-APC, cold-stored apheresis-derived platelet concentrates; PB, peripheral blood; RT, room temperature; SEM, standard error of mean; TRAP-6, thrombin receptor activating peptide; VASP, vasodilator-stimulated phosphoprotein.

    Article Snippet: Rabbit polyclonal anti-P2Y1, anti-P2Y12, and anti-P2X1 antibodies were from Alomone Labs (Jerusalem, Israel).

    Techniques: Expressing, Fluorescence, Flow Cytometry, Derivative Assay

    A, Relative expression of P2X 7 mRNA detected by qPCR. B, SDS-PAGE electrophoresis of P2X 7 receptor protein. C, Relative expression of P2X 7 receptor protein; ** P < 0.01, versus Ctrl group; n = 5.

    Journal: PLoS ONE

    Article Title: Effect of naringin on gp120-induced injury mediated by P2X 7 receptors in rat primary cultured microglia

    doi: 10.1371/journal.pone.0183688

    Figure Lengend Snippet: A, Relative expression of P2X 7 mRNA detected by qPCR. B, SDS-PAGE electrophoresis of P2X 7 receptor protein. C, Relative expression of P2X 7 receptor protein; ** P < 0.01, versus Ctrl group; n = 5.

    Article Snippet: The primary antibodies were as follows: rabbit polyclonal anti-P2X 7 (Alomone, Israel; 1:400 dilution), and β-actin (Beijing Zhongshan Biotech Co., 1:800 dilution).

    Techniques: Expressing, SDS Page, Electrophoresis

    A, SDS-PAGE electrophoresis of P2X 7 receptor proteins in primary cultured microglia. B, Relative expression level of P2X 7 receptor protein in primary cultured microglia; ** P < 0.01, versus Ctrl group; ## P < 0.01, versus gp120 group; n = 5.

    Journal: PLoS ONE

    Article Title: Effect of naringin on gp120-induced injury mediated by P2X 7 receptors in rat primary cultured microglia

    doi: 10.1371/journal.pone.0183688

    Figure Lengend Snippet: A, SDS-PAGE electrophoresis of P2X 7 receptor proteins in primary cultured microglia. B, Relative expression level of P2X 7 receptor protein in primary cultured microglia; ** P < 0.01, versus Ctrl group; ## P < 0.01, versus gp120 group; n = 5.

    Article Snippet: The primary antibodies were as follows: rabbit polyclonal anti-P2X 7 (Alomone, Israel; 1:400 dilution), and β-actin (Beijing Zhongshan Biotech Co., 1:800 dilution).

    Techniques: SDS Page, Electrophoresis, Cell Culture, Expressing

    A, Typical fluorescence image of primary cultured microglia; green signal represents CD11b staining with FITC; red signal indicates P2X 7 staining with TRITC; Merge represents the P2X 7 and CD11b double staining image; arrows represent microglia with co-expression of P2X 7 receptor and CD11b. B, Co-expression values of CD11b and P2X 7 receptor in each group; ** P < 0.01, versus Ctrl group; ## P < 0.01, versus gp120 group; n = 3.

    Journal: PLoS ONE

    Article Title: Effect of naringin on gp120-induced injury mediated by P2X 7 receptors in rat primary cultured microglia

    doi: 10.1371/journal.pone.0183688

    Figure Lengend Snippet: A, Typical fluorescence image of primary cultured microglia; green signal represents CD11b staining with FITC; red signal indicates P2X 7 staining with TRITC; Merge represents the P2X 7 and CD11b double staining image; arrows represent microglia with co-expression of P2X 7 receptor and CD11b. B, Co-expression values of CD11b and P2X 7 receptor in each group; ** P < 0.01, versus Ctrl group; ## P < 0.01, versus gp120 group; n = 3.

    Article Snippet: The primary antibodies were as follows: rabbit polyclonal anti-P2X 7 (Alomone, Israel; 1:400 dilution), and β-actin (Beijing Zhongshan Biotech Co., 1:800 dilution).

    Techniques: Fluorescence, Cell Culture, Staining, Double Staining, Expressing