anti adenosine a2b receptor  (Alomone Labs)


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    Structured Review

    Alomone Labs anti adenosine a2b receptor
    CAR T cell enhancement by BAY 60-6583 in CD133 CAR T cells seems not to be dependent on the adenosine <t>A2b</t> receptor. (A, B) Blocking of the adenosine A2b receptor cannot inhibit the enhanced antitumor activities induced by BAY 60-6583. In the anti-CD133 CAR T co-culture system (E:T ratio of 4:1), the adenosine A2b receptor was blocked by 1 μM PSB603 1 h before 10 μM BAY 60-6583 or 1 μM NECA was added. 24 h later, TNF-α production (A) and tumor lysis (B) were analyzed (C) The expression of the adenosine A2b receptor on conventional (WT) and A2b receptor knockout (4 + 6) anti-CD133 CAR T cells was determined by Western blot. Data from a representative experiment of n = 4 experiments (D, E) BAY 60-6583-induced enhancement of CAR T cell-mediated antitumor activities were not influenced by adenosine A2b receptor deficiency. Conventional and A2b receptor knockout anti-CD133 CAR T cells were co-cultured with U251-CD133OE luc cells at an E:T ratio of 4:1. After treatment with vehicle control or BAY 60-6583 for 24 h, cytokine secretion in the supernatant was detected (D) ; 48 h later, cytotoxicity was assessed (E) . * p
    Anti Adenosine A2b Receptor, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti adenosine a2b receptor/product/Alomone Labs
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti adenosine a2b receptor - by Bioz Stars, 2022-01
    94/100 stars

    Images

    1) Product Images from "BAY 60-6583 Enhances the Antitumor Function of Chimeric Antigen Receptor-Modified T Cells Independent of the Adenosine A2b Receptor"

    Article Title: BAY 60-6583 Enhances the Antitumor Function of Chimeric Antigen Receptor-Modified T Cells Independent of the Adenosine A2b Receptor

    Journal: Frontiers in Pharmacology

    doi: 10.3389/fphar.2021.619800

    CAR T cell enhancement by BAY 60-6583 in CD133 CAR T cells seems not to be dependent on the adenosine A2b receptor. (A, B) Blocking of the adenosine A2b receptor cannot inhibit the enhanced antitumor activities induced by BAY 60-6583. In the anti-CD133 CAR T co-culture system (E:T ratio of 4:1), the adenosine A2b receptor was blocked by 1 μM PSB603 1 h before 10 μM BAY 60-6583 or 1 μM NECA was added. 24 h later, TNF-α production (A) and tumor lysis (B) were analyzed (C) The expression of the adenosine A2b receptor on conventional (WT) and A2b receptor knockout (4 + 6) anti-CD133 CAR T cells was determined by Western blot. Data from a representative experiment of n = 4 experiments (D, E) BAY 60-6583-induced enhancement of CAR T cell-mediated antitumor activities were not influenced by adenosine A2b receptor deficiency. Conventional and A2b receptor knockout anti-CD133 CAR T cells were co-cultured with U251-CD133OE luc cells at an E:T ratio of 4:1. After treatment with vehicle control or BAY 60-6583 for 24 h, cytokine secretion in the supernatant was detected (D) ; 48 h later, cytotoxicity was assessed (E) . * p
    Figure Legend Snippet: CAR T cell enhancement by BAY 60-6583 in CD133 CAR T cells seems not to be dependent on the adenosine A2b receptor. (A, B) Blocking of the adenosine A2b receptor cannot inhibit the enhanced antitumor activities induced by BAY 60-6583. In the anti-CD133 CAR T co-culture system (E:T ratio of 4:1), the adenosine A2b receptor was blocked by 1 μM PSB603 1 h before 10 μM BAY 60-6583 or 1 μM NECA was added. 24 h later, TNF-α production (A) and tumor lysis (B) were analyzed (C) The expression of the adenosine A2b receptor on conventional (WT) and A2b receptor knockout (4 + 6) anti-CD133 CAR T cells was determined by Western blot. Data from a representative experiment of n = 4 experiments (D, E) BAY 60-6583-induced enhancement of CAR T cell-mediated antitumor activities were not influenced by adenosine A2b receptor deficiency. Conventional and A2b receptor knockout anti-CD133 CAR T cells were co-cultured with U251-CD133OE luc cells at an E:T ratio of 4:1. After treatment with vehicle control or BAY 60-6583 for 24 h, cytokine secretion in the supernatant was detected (D) ; 48 h later, cytotoxicity was assessed (E) . * p

    Techniques Used: Blocking Assay, Co-Culture Assay, Lysis, Expressing, Knock-Out, Western Blot, Cell Culture

    2) Product Images from "The Cholangiocyte Adenosine–IL-6 Axis Regulates Survival During Biliary Cirrhosis"

    Article Title: The Cholangiocyte Adenosine–IL-6 Axis Regulates Survival During Biliary Cirrhosis

    Journal: Gene Expression

    doi: 10.3727/105221617X15042723767876

    Bile duct staining with CK19 (A–C) and cell proliferation analysis by Ki-67 staining (D, E) were performed 2 weeks after BDL mice. A group of mice received recombinant mouse IL-6 (IL-6) or the vehicle only (vehicle) for both sham and BDL surgeries. (A) Representative CK19 staining image for each group. Scale bar: 50 μm. (D, E) Count of CK19 + duct (D; with visible lumen) and nonduct (E; no lumen) per portal area in a blinded manner. The following were the number of animals per group: C57BL/6 sham (7), C57BL/6 sham + IL-6 (3), C57BL/6 BDL (9), C57BL/6 BDL + IL-6 (3), A2bAR −/− sham (7), A2bAR −/− sham + IL-6 (3), A2bAR −/− BDL (13), and A2bAR −/− BDL + IL-6 (3). Between 3 and 5 100× magnification fields were count per animal. #Significant when compared to C57BL/6 BDL vehicle; *significant when compared to A2bAR −/− BDL vehicle. (D) Representative image of Ki-67 staining for BDL-operated animal with or without recmIL-6 injection. Scale bar: 50 μm. (E) Analysis of the percent of Ki-67 + pixels on five different 100× magnification fields for three animals per group. #Significant when compared to C57BL/6 BDL vehicle; *significant when compared to C57BL/6 BDL IL-6.
    Figure Legend Snippet: Bile duct staining with CK19 (A–C) and cell proliferation analysis by Ki-67 staining (D, E) were performed 2 weeks after BDL mice. A group of mice received recombinant mouse IL-6 (IL-6) or the vehicle only (vehicle) for both sham and BDL surgeries. (A) Representative CK19 staining image for each group. Scale bar: 50 μm. (D, E) Count of CK19 + duct (D; with visible lumen) and nonduct (E; no lumen) per portal area in a blinded manner. The following were the number of animals per group: C57BL/6 sham (7), C57BL/6 sham + IL-6 (3), C57BL/6 BDL (9), C57BL/6 BDL + IL-6 (3), A2bAR −/− sham (7), A2bAR −/− sham + IL-6 (3), A2bAR −/− BDL (13), and A2bAR −/− BDL + IL-6 (3). Between 3 and 5 100× magnification fields were count per animal. #Significant when compared to C57BL/6 BDL vehicle; *significant when compared to A2bAR −/− BDL vehicle. (D) Representative image of Ki-67 staining for BDL-operated animal with or without recmIL-6 injection. Scale bar: 50 μm. (E) Analysis of the percent of Ki-67 + pixels on five different 100× magnification fields for three animals per group. #Significant when compared to C57BL/6 BDL vehicle; *significant when compared to C57BL/6 BDL IL-6.

    Techniques Used: Staining, Mouse Assay, Recombinant, Injection

    Second messenger alterations and control of IL-6 release mediated by adenosine/A2bAR. Intracellular cAMP concentrations (A, B) were analyzed by ELISA. H69 cells were stimulated with 100 μM adenosine for 2 h in 96-well plates (5,000 cells per well) in H69 complete media in the presence of A2bAR antagonist (A) MRS-1754 (1 μM; n = 3, performed in triplicates) or (B) 72 h after transfection with an A2bAR-specific siRNA ( n = 4, performed in triplicate). #Significant when compared to the untreated cells; *significant when compared to vehicle (A) or nontarget siRNA (B). (C, D) Intracellular Ca 2+ mobilization was observed by confocal microscopy. Glass coverslip-plated, fluo-4/AM-loaded H69 cells were perifused with HEPES buffer alone or containing 500 μM adenosine. ATP (100 μM) was used as a positive control. Changes in fluorescence intensity were recorded with a Zeiss 510 confocal microscope. (C) Relative fluorescence intensity of individual cells; 29 individual cells were analyzed. (D) Representative image of the dye intensity variation during the course of the experiment. Scale bar: 50 μm. (E) H69 cells were stimulated by 100 μM of adenosine for 2 h in six-well plates (150,000 cells per well) in H69 complete media. RNA was extracted, and IL-6 mRNA expression was determined by qPCR. Cells were preincubated with BAPTA/AM (50 μM) or cAMPs-RP (100 μM) 30 min prior to the stimulation ( n = 3 for cAMPs-RP and n = 7 for BAPTA/AM). (F) H69 cells were stimulated by 100 μM adenosine for 4 h in 48-well plates (30,000 cells per well) in H69 complete media. Cells were preincubated with BAPTA/AM (50 μM) or cAMPs-RP (100 μM) 30 min prior to the stimulation. Supernatants were collected and centrifuged, and IL-6 content was analyzed by ELISA ( n = 3, performed in triplicate). #Significant when compared to unstimulated cells; *significant when compared to vehicle. (G) H69 cells were stimulated by 100 μM of 8-pCPT-2-O-Me-cAMP-AM (8-pCPT) or adenosine for 4 h in 48-well plates (30,000 cells per well) in H69 complete media. Supernatants were collected and centrifuged, and IL-6 content was analyzed by ELISA ( n = 3, performed in triplicate).
    Figure Legend Snippet: Second messenger alterations and control of IL-6 release mediated by adenosine/A2bAR. Intracellular cAMP concentrations (A, B) were analyzed by ELISA. H69 cells were stimulated with 100 μM adenosine for 2 h in 96-well plates (5,000 cells per well) in H69 complete media in the presence of A2bAR antagonist (A) MRS-1754 (1 μM; n = 3, performed in triplicates) or (B) 72 h after transfection with an A2bAR-specific siRNA ( n = 4, performed in triplicate). #Significant when compared to the untreated cells; *significant when compared to vehicle (A) or nontarget siRNA (B). (C, D) Intracellular Ca 2+ mobilization was observed by confocal microscopy. Glass coverslip-plated, fluo-4/AM-loaded H69 cells were perifused with HEPES buffer alone or containing 500 μM adenosine. ATP (100 μM) was used as a positive control. Changes in fluorescence intensity were recorded with a Zeiss 510 confocal microscope. (C) Relative fluorescence intensity of individual cells; 29 individual cells were analyzed. (D) Representative image of the dye intensity variation during the course of the experiment. Scale bar: 50 μm. (E) H69 cells were stimulated by 100 μM of adenosine for 2 h in six-well plates (150,000 cells per well) in H69 complete media. RNA was extracted, and IL-6 mRNA expression was determined by qPCR. Cells were preincubated with BAPTA/AM (50 μM) or cAMPs-RP (100 μM) 30 min prior to the stimulation ( n = 3 for cAMPs-RP and n = 7 for BAPTA/AM). (F) H69 cells were stimulated by 100 μM adenosine for 4 h in 48-well plates (30,000 cells per well) in H69 complete media. Cells were preincubated with BAPTA/AM (50 μM) or cAMPs-RP (100 μM) 30 min prior to the stimulation. Supernatants were collected and centrifuged, and IL-6 content was analyzed by ELISA ( n = 3, performed in triplicate). #Significant when compared to unstimulated cells; *significant when compared to vehicle. (G) H69 cells were stimulated by 100 μM of 8-pCPT-2-O-Me-cAMP-AM (8-pCPT) or adenosine for 4 h in 48-well plates (30,000 cells per well) in H69 complete media. Supernatants were collected and centrifuged, and IL-6 content was analyzed by ELISA ( n = 3, performed in triplicate).

    Techniques Used: Enzyme-linked Immunosorbent Assay, Transfection, Confocal Microscopy, Positive Control, Fluorescence, Microscopy, Expressing, Real-time Polymerase Chain Reaction

    Effect of A2bAR knockout on survival and fibrosis after bile duct ligation (BDL). BDL was performed on 10- to 12-week-old male C57BL/6 or A2bAR −/− mice. (A–D) A group of mice received weekly subcutaneous injection of recombinant mouse IL-6 (IL-6; n = 13 for both background), and the other group received vehicle only ( n = 8 for both background). (A) Kaplan–Meier survival graph. The black line represents control C57BL/6 mice, and the blue line corresponds to A2bAR −/− mice. Full lines are vehicle only, and dotted lines represent mice that received recmIL-6 injections. *Significant when compared to C57BL/6 mice; #significant when compared to vehicle injection. (B) Mason trichrome staining representative of each group at the median time of survival. Scale bar: 100 μm. (C) METAVIR fibrosis score analysis of the Mason trichrome staining performed in a blinded manner. (D) Bile infarcts were counted on a blinded manner on low-power field hematoxylin and eosin staining of each individual mouse. (E–H) BDL or sham operation was performed on 10- to 12-week-old male C57BL/6 ( n = 9 and 7, respectively) or A2bAR −/− mice ( n = 13 and 7, respectively). Mice were sacrificed 2 weeks after surgery. (E) Representative picture of hematoxylin and eosin (H E), Pico Sirius red (PSR), and Mason trichrome staining for each group of mice. Scale bar: 100 μm. (F) METAVIR fibrosis score analysis of the Mason trichrome staining performed in a blinded manner. (G) Total liver RNA was extracted, and relative IL-6 mRNA was assessed by qPCR. GAPDH -specific probe was used as control. C57BL/6 and A2bAR −/− sham operated levels were, respectively, used as reference for the BDL group. (H) Serum was obtained from sham and BDL-operated mice, and total serum IL-6 was assessed by ELISA.
    Figure Legend Snippet: Effect of A2bAR knockout on survival and fibrosis after bile duct ligation (BDL). BDL was performed on 10- to 12-week-old male C57BL/6 or A2bAR −/− mice. (A–D) A group of mice received weekly subcutaneous injection of recombinant mouse IL-6 (IL-6; n = 13 for both background), and the other group received vehicle only ( n = 8 for both background). (A) Kaplan–Meier survival graph. The black line represents control C57BL/6 mice, and the blue line corresponds to A2bAR −/− mice. Full lines are vehicle only, and dotted lines represent mice that received recmIL-6 injections. *Significant when compared to C57BL/6 mice; #significant when compared to vehicle injection. (B) Mason trichrome staining representative of each group at the median time of survival. Scale bar: 100 μm. (C) METAVIR fibrosis score analysis of the Mason trichrome staining performed in a blinded manner. (D) Bile infarcts were counted on a blinded manner on low-power field hematoxylin and eosin staining of each individual mouse. (E–H) BDL or sham operation was performed on 10- to 12-week-old male C57BL/6 ( n = 9 and 7, respectively) or A2bAR −/− mice ( n = 13 and 7, respectively). Mice were sacrificed 2 weeks after surgery. (E) Representative picture of hematoxylin and eosin (H E), Pico Sirius red (PSR), and Mason trichrome staining for each group of mice. Scale bar: 100 μm. (F) METAVIR fibrosis score analysis of the Mason trichrome staining performed in a blinded manner. (G) Total liver RNA was extracted, and relative IL-6 mRNA was assessed by qPCR. GAPDH -specific probe was used as control. C57BL/6 and A2bAR −/− sham operated levels were, respectively, used as reference for the BDL group. (H) Serum was obtained from sham and BDL-operated mice, and total serum IL-6 was assessed by ELISA.

    Techniques Used: Knock-Out, Ligation, Mouse Assay, Injection, Recombinant, Staining, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

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    Alomone Labs anti adenosine a2b receptor
    CAR T cell enhancement by BAY 60-6583 in CD133 CAR T cells seems not to be dependent on the adenosine <t>A2b</t> receptor. (A, B) Blocking of the adenosine A2b receptor cannot inhibit the enhanced antitumor activities induced by BAY 60-6583. In the anti-CD133 CAR T co-culture system (E:T ratio of 4:1), the adenosine A2b receptor was blocked by 1 μM PSB603 1 h before 10 μM BAY 60-6583 or 1 μM NECA was added. 24 h later, TNF-α production (A) and tumor lysis (B) were analyzed (C) The expression of the adenosine A2b receptor on conventional (WT) and A2b receptor knockout (4 + 6) anti-CD133 CAR T cells was determined by Western blot. Data from a representative experiment of n = 4 experiments (D, E) BAY 60-6583-induced enhancement of CAR T cell-mediated antitumor activities were not influenced by adenosine A2b receptor deficiency. Conventional and A2b receptor knockout anti-CD133 CAR T cells were co-cultured with U251-CD133OE luc cells at an E:T ratio of 4:1. After treatment with vehicle control or BAY 60-6583 for 24 h, cytokine secretion in the supernatant was detected (D) ; 48 h later, cytotoxicity was assessed (E) . * p
    Anti Adenosine A2b Receptor, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti adenosine a2b receptor/product/Alomone Labs
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti adenosine a2b receptor - by Bioz Stars, 2022-01
    94/100 stars
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    CAR T cell enhancement by BAY 60-6583 in CD133 CAR T cells seems not to be dependent on the adenosine A2b receptor. (A, B) Blocking of the adenosine A2b receptor cannot inhibit the enhanced antitumor activities induced by BAY 60-6583. In the anti-CD133 CAR T co-culture system (E:T ratio of 4:1), the adenosine A2b receptor was blocked by 1 μM PSB603 1 h before 10 μM BAY 60-6583 or 1 μM NECA was added. 24 h later, TNF-α production (A) and tumor lysis (B) were analyzed (C) The expression of the adenosine A2b receptor on conventional (WT) and A2b receptor knockout (4 + 6) anti-CD133 CAR T cells was determined by Western blot. Data from a representative experiment of n = 4 experiments (D, E) BAY 60-6583-induced enhancement of CAR T cell-mediated antitumor activities were not influenced by adenosine A2b receptor deficiency. Conventional and A2b receptor knockout anti-CD133 CAR T cells were co-cultured with U251-CD133OE luc cells at an E:T ratio of 4:1. After treatment with vehicle control or BAY 60-6583 for 24 h, cytokine secretion in the supernatant was detected (D) ; 48 h later, cytotoxicity was assessed (E) . * p

    Journal: Frontiers in Pharmacology

    Article Title: BAY 60-6583 Enhances the Antitumor Function of Chimeric Antigen Receptor-Modified T Cells Independent of the Adenosine A2b Receptor

    doi: 10.3389/fphar.2021.619800

    Figure Lengend Snippet: CAR T cell enhancement by BAY 60-6583 in CD133 CAR T cells seems not to be dependent on the adenosine A2b receptor. (A, B) Blocking of the adenosine A2b receptor cannot inhibit the enhanced antitumor activities induced by BAY 60-6583. In the anti-CD133 CAR T co-culture system (E:T ratio of 4:1), the adenosine A2b receptor was blocked by 1 μM PSB603 1 h before 10 μM BAY 60-6583 or 1 μM NECA was added. 24 h later, TNF-α production (A) and tumor lysis (B) were analyzed (C) The expression of the adenosine A2b receptor on conventional (WT) and A2b receptor knockout (4 + 6) anti-CD133 CAR T cells was determined by Western blot. Data from a representative experiment of n = 4 experiments (D, E) BAY 60-6583-induced enhancement of CAR T cell-mediated antitumor activities were not influenced by adenosine A2b receptor deficiency. Conventional and A2b receptor knockout anti-CD133 CAR T cells were co-cultured with U251-CD133OE luc cells at an E:T ratio of 4:1. After treatment with vehicle control or BAY 60-6583 for 24 h, cytokine secretion in the supernatant was detected (D) ; 48 h later, cytotoxicity was assessed (E) . * p

    Article Snippet: After blocking with 5% milk for 1 h, the membranes were incubated with anti-adenosine A2b receptor (AAR-003, alomone labs) or anti-GAPDH (sc-32233, Santa Cruz Biotechnology) antibodies at 4°C overnight.

    Techniques: Blocking Assay, Co-Culture Assay, Lysis, Expressing, Knock-Out, Western Blot, Cell Culture

    Bile duct staining with CK19 (A–C) and cell proliferation analysis by Ki-67 staining (D, E) were performed 2 weeks after BDL mice. A group of mice received recombinant mouse IL-6 (IL-6) or the vehicle only (vehicle) for both sham and BDL surgeries. (A) Representative CK19 staining image for each group. Scale bar: 50 μm. (D, E) Count of CK19 + duct (D; with visible lumen) and nonduct (E; no lumen) per portal area in a blinded manner. The following were the number of animals per group: C57BL/6 sham (7), C57BL/6 sham + IL-6 (3), C57BL/6 BDL (9), C57BL/6 BDL + IL-6 (3), A2bAR −/− sham (7), A2bAR −/− sham + IL-6 (3), A2bAR −/− BDL (13), and A2bAR −/− BDL + IL-6 (3). Between 3 and 5 100× magnification fields were count per animal. #Significant when compared to C57BL/6 BDL vehicle; *significant when compared to A2bAR −/− BDL vehicle. (D) Representative image of Ki-67 staining for BDL-operated animal with or without recmIL-6 injection. Scale bar: 50 μm. (E) Analysis of the percent of Ki-67 + pixels on five different 100× magnification fields for three animals per group. #Significant when compared to C57BL/6 BDL vehicle; *significant when compared to C57BL/6 BDL IL-6.

    Journal: Gene Expression

    Article Title: The Cholangiocyte Adenosine–IL-6 Axis Regulates Survival During Biliary Cirrhosis

    doi: 10.3727/105221617X15042723767876

    Figure Lengend Snippet: Bile duct staining with CK19 (A–C) and cell proliferation analysis by Ki-67 staining (D, E) were performed 2 weeks after BDL mice. A group of mice received recombinant mouse IL-6 (IL-6) or the vehicle only (vehicle) for both sham and BDL surgeries. (A) Representative CK19 staining image for each group. Scale bar: 50 μm. (D, E) Count of CK19 + duct (D; with visible lumen) and nonduct (E; no lumen) per portal area in a blinded manner. The following were the number of animals per group: C57BL/6 sham (7), C57BL/6 sham + IL-6 (3), C57BL/6 BDL (9), C57BL/6 BDL + IL-6 (3), A2bAR −/− sham (7), A2bAR −/− sham + IL-6 (3), A2bAR −/− BDL (13), and A2bAR −/− BDL + IL-6 (3). Between 3 and 5 100× magnification fields were count per animal. #Significant when compared to C57BL/6 BDL vehicle; *significant when compared to A2bAR −/− BDL vehicle. (D) Representative image of Ki-67 staining for BDL-operated animal with or without recmIL-6 injection. Scale bar: 50 μm. (E) Analysis of the percent of Ki-67 + pixels on five different 100× magnification fields for three animals per group. #Significant when compared to C57BL/6 BDL vehicle; *significant when compared to C57BL/6 BDL IL-6.

    Article Snippet: Antibodies used in this study are as follows: polyclonal rabbit anti-Ki-67 antibody (Abcam, Cambridge, MA, USA), polyclonal rabbit anti-A2bAR antibody (Alomone Labs, Jerusalem, Israel), monoclonal rat anti-mouse neutrophil antibody (NIMP-R14; Abcam), monoclonal rat anti-mouse F4/80 (Cl:A3-1; Bio-Rad Laboratories, Hercules, CA), and monoclonal rabbit anti-CD3 (SP7; Abcam).

    Techniques: Staining, Mouse Assay, Recombinant, Injection

    Second messenger alterations and control of IL-6 release mediated by adenosine/A2bAR. Intracellular cAMP concentrations (A, B) were analyzed by ELISA. H69 cells were stimulated with 100 μM adenosine for 2 h in 96-well plates (5,000 cells per well) in H69 complete media in the presence of A2bAR antagonist (A) MRS-1754 (1 μM; n = 3, performed in triplicates) or (B) 72 h after transfection with an A2bAR-specific siRNA ( n = 4, performed in triplicate). #Significant when compared to the untreated cells; *significant when compared to vehicle (A) or nontarget siRNA (B). (C, D) Intracellular Ca 2+ mobilization was observed by confocal microscopy. Glass coverslip-plated, fluo-4/AM-loaded H69 cells were perifused with HEPES buffer alone or containing 500 μM adenosine. ATP (100 μM) was used as a positive control. Changes in fluorescence intensity were recorded with a Zeiss 510 confocal microscope. (C) Relative fluorescence intensity of individual cells; 29 individual cells were analyzed. (D) Representative image of the dye intensity variation during the course of the experiment. Scale bar: 50 μm. (E) H69 cells were stimulated by 100 μM of adenosine for 2 h in six-well plates (150,000 cells per well) in H69 complete media. RNA was extracted, and IL-6 mRNA expression was determined by qPCR. Cells were preincubated with BAPTA/AM (50 μM) or cAMPs-RP (100 μM) 30 min prior to the stimulation ( n = 3 for cAMPs-RP and n = 7 for BAPTA/AM). (F) H69 cells were stimulated by 100 μM adenosine for 4 h in 48-well plates (30,000 cells per well) in H69 complete media. Cells were preincubated with BAPTA/AM (50 μM) or cAMPs-RP (100 μM) 30 min prior to the stimulation. Supernatants were collected and centrifuged, and IL-6 content was analyzed by ELISA ( n = 3, performed in triplicate). #Significant when compared to unstimulated cells; *significant when compared to vehicle. (G) H69 cells were stimulated by 100 μM of 8-pCPT-2-O-Me-cAMP-AM (8-pCPT) or adenosine for 4 h in 48-well plates (30,000 cells per well) in H69 complete media. Supernatants were collected and centrifuged, and IL-6 content was analyzed by ELISA ( n = 3, performed in triplicate).

    Journal: Gene Expression

    Article Title: The Cholangiocyte Adenosine–IL-6 Axis Regulates Survival During Biliary Cirrhosis

    doi: 10.3727/105221617X15042723767876

    Figure Lengend Snippet: Second messenger alterations and control of IL-6 release mediated by adenosine/A2bAR. Intracellular cAMP concentrations (A, B) were analyzed by ELISA. H69 cells were stimulated with 100 μM adenosine for 2 h in 96-well plates (5,000 cells per well) in H69 complete media in the presence of A2bAR antagonist (A) MRS-1754 (1 μM; n = 3, performed in triplicates) or (B) 72 h after transfection with an A2bAR-specific siRNA ( n = 4, performed in triplicate). #Significant when compared to the untreated cells; *significant when compared to vehicle (A) or nontarget siRNA (B). (C, D) Intracellular Ca 2+ mobilization was observed by confocal microscopy. Glass coverslip-plated, fluo-4/AM-loaded H69 cells were perifused with HEPES buffer alone or containing 500 μM adenosine. ATP (100 μM) was used as a positive control. Changes in fluorescence intensity were recorded with a Zeiss 510 confocal microscope. (C) Relative fluorescence intensity of individual cells; 29 individual cells were analyzed. (D) Representative image of the dye intensity variation during the course of the experiment. Scale bar: 50 μm. (E) H69 cells were stimulated by 100 μM of adenosine for 2 h in six-well plates (150,000 cells per well) in H69 complete media. RNA was extracted, and IL-6 mRNA expression was determined by qPCR. Cells were preincubated with BAPTA/AM (50 μM) or cAMPs-RP (100 μM) 30 min prior to the stimulation ( n = 3 for cAMPs-RP and n = 7 for BAPTA/AM). (F) H69 cells were stimulated by 100 μM adenosine for 4 h in 48-well plates (30,000 cells per well) in H69 complete media. Cells were preincubated with BAPTA/AM (50 μM) or cAMPs-RP (100 μM) 30 min prior to the stimulation. Supernatants were collected and centrifuged, and IL-6 content was analyzed by ELISA ( n = 3, performed in triplicate). #Significant when compared to unstimulated cells; *significant when compared to vehicle. (G) H69 cells were stimulated by 100 μM of 8-pCPT-2-O-Me-cAMP-AM (8-pCPT) or adenosine for 4 h in 48-well plates (30,000 cells per well) in H69 complete media. Supernatants were collected and centrifuged, and IL-6 content was analyzed by ELISA ( n = 3, performed in triplicate).

    Article Snippet: Antibodies used in this study are as follows: polyclonal rabbit anti-Ki-67 antibody (Abcam, Cambridge, MA, USA), polyclonal rabbit anti-A2bAR antibody (Alomone Labs, Jerusalem, Israel), monoclonal rat anti-mouse neutrophil antibody (NIMP-R14; Abcam), monoclonal rat anti-mouse F4/80 (Cl:A3-1; Bio-Rad Laboratories, Hercules, CA), and monoclonal rabbit anti-CD3 (SP7; Abcam).

    Techniques: Enzyme-linked Immunosorbent Assay, Transfection, Confocal Microscopy, Positive Control, Fluorescence, Microscopy, Expressing, Real-time Polymerase Chain Reaction

    Effect of A2bAR knockout on survival and fibrosis after bile duct ligation (BDL). BDL was performed on 10- to 12-week-old male C57BL/6 or A2bAR −/− mice. (A–D) A group of mice received weekly subcutaneous injection of recombinant mouse IL-6 (IL-6; n = 13 for both background), and the other group received vehicle only ( n = 8 for both background). (A) Kaplan–Meier survival graph. The black line represents control C57BL/6 mice, and the blue line corresponds to A2bAR −/− mice. Full lines are vehicle only, and dotted lines represent mice that received recmIL-6 injections. *Significant when compared to C57BL/6 mice; #significant when compared to vehicle injection. (B) Mason trichrome staining representative of each group at the median time of survival. Scale bar: 100 μm. (C) METAVIR fibrosis score analysis of the Mason trichrome staining performed in a blinded manner. (D) Bile infarcts were counted on a blinded manner on low-power field hematoxylin and eosin staining of each individual mouse. (E–H) BDL or sham operation was performed on 10- to 12-week-old male C57BL/6 ( n = 9 and 7, respectively) or A2bAR −/− mice ( n = 13 and 7, respectively). Mice were sacrificed 2 weeks after surgery. (E) Representative picture of hematoxylin and eosin (H E), Pico Sirius red (PSR), and Mason trichrome staining for each group of mice. Scale bar: 100 μm. (F) METAVIR fibrosis score analysis of the Mason trichrome staining performed in a blinded manner. (G) Total liver RNA was extracted, and relative IL-6 mRNA was assessed by qPCR. GAPDH -specific probe was used as control. C57BL/6 and A2bAR −/− sham operated levels were, respectively, used as reference for the BDL group. (H) Serum was obtained from sham and BDL-operated mice, and total serum IL-6 was assessed by ELISA.

    Journal: Gene Expression

    Article Title: The Cholangiocyte Adenosine–IL-6 Axis Regulates Survival During Biliary Cirrhosis

    doi: 10.3727/105221617X15042723767876

    Figure Lengend Snippet: Effect of A2bAR knockout on survival and fibrosis after bile duct ligation (BDL). BDL was performed on 10- to 12-week-old male C57BL/6 or A2bAR −/− mice. (A–D) A group of mice received weekly subcutaneous injection of recombinant mouse IL-6 (IL-6; n = 13 for both background), and the other group received vehicle only ( n = 8 for both background). (A) Kaplan–Meier survival graph. The black line represents control C57BL/6 mice, and the blue line corresponds to A2bAR −/− mice. Full lines are vehicle only, and dotted lines represent mice that received recmIL-6 injections. *Significant when compared to C57BL/6 mice; #significant when compared to vehicle injection. (B) Mason trichrome staining representative of each group at the median time of survival. Scale bar: 100 μm. (C) METAVIR fibrosis score analysis of the Mason trichrome staining performed in a blinded manner. (D) Bile infarcts were counted on a blinded manner on low-power field hematoxylin and eosin staining of each individual mouse. (E–H) BDL or sham operation was performed on 10- to 12-week-old male C57BL/6 ( n = 9 and 7, respectively) or A2bAR −/− mice ( n = 13 and 7, respectively). Mice were sacrificed 2 weeks after surgery. (E) Representative picture of hematoxylin and eosin (H E), Pico Sirius red (PSR), and Mason trichrome staining for each group of mice. Scale bar: 100 μm. (F) METAVIR fibrosis score analysis of the Mason trichrome staining performed in a blinded manner. (G) Total liver RNA was extracted, and relative IL-6 mRNA was assessed by qPCR. GAPDH -specific probe was used as control. C57BL/6 and A2bAR −/− sham operated levels were, respectively, used as reference for the BDL group. (H) Serum was obtained from sham and BDL-operated mice, and total serum IL-6 was assessed by ELISA.

    Article Snippet: Antibodies used in this study are as follows: polyclonal rabbit anti-Ki-67 antibody (Abcam, Cambridge, MA, USA), polyclonal rabbit anti-A2bAR antibody (Alomone Labs, Jerusalem, Israel), monoclonal rat anti-mouse neutrophil antibody (NIMP-R14; Abcam), monoclonal rat anti-mouse F4/80 (Cl:A3-1; Bio-Rad Laboratories, Hercules, CA), and monoclonal rabbit anti-CD3 (SP7; Abcam).

    Techniques: Knock-Out, Ligation, Mouse Assay, Injection, Recombinant, Staining, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay