anti p2x7 receptor extracellular fitc antibody  (Alomone Labs)

Journal: Journal of Inflammation Research

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

doi: 10.2147/JIR.S351660

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

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

Techniques:

Journal: Journal of Inflammation Research

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

doi: 10.2147/JIR.S351660

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

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

Techniques: Expressing, Flow Cytometry

Journal: Journal of Inflammation Research

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

doi: 10.2147/JIR.S351660

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

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

Techniques: Quantitative RT-PCR

Journal: Journal of Inflammation Research

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

doi: 10.2147/JIR.S351660

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

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

Techniques: Expressing

Journal: Journal of Inflammation Research

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

doi: 10.2147/JIR.S351660

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

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

Techniques: Expressing

Journal: Journal of Inflammation Research

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

doi: 10.2147/JIR.S351660

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

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

Techniques:

Journal: Cell Reports

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

doi: 10.1016/j.celrep.2019.03.011

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

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

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

Journal: Cell Reports

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

doi: 10.1016/j.celrep.2019.03.011

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

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

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

Journal: Cell Reports

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

doi: 10.1016/j.celrep.2019.03.011

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

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

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

Journal: Cell Reports

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

doi: 10.1016/j.celrep.2019.03.011

Figure Lengend Snippet:

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

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

Journal: Cell Reports

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

doi: 10.1016/j.celrep.2019.03.011

Figure Lengend Snippet:

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

Techniques: