Journal: eLife

Article Title: Effector membrane translocation biosensors reveal G protein and βarrestin coupling profiles of 100 therapeutically relevant GPCRs

doi: 10.7554/eLife.74101

Figure Lengend Snippet: ( A ) Pharmacological validation of the Gα i/o activation sensor. HEK293 cells were transfected with the D 2 receptor and the Gα i/o family-specific sensor, along with each Gα i/o subunit. Concentration-response curve using the Gα i/o activation sensor, in the presence or absence of UBO-QIC ( left ) or PTX ( right ) inhibitors. Insets ; E max values determined from concentration-response curves of inhibitor-pretreated cells. ( B ) Pharmacological validation of the Gα q/11 activation sensor. HEK293 cells were transfected with the GnRH receptor and the Gα q/11 family-specific sensor, along with each Gα q/11 subunit. Concentration-response curve using Gα q/11 activation sensor, in the presence or absence of UBO-QIC ( left ) or PTX ( right ) inhibitors. Insets ; E max values determined from dose-response curves of inhibitor-pretreated cells. ( C ) Validation of the Gα 12/13 activation sensor. Cells were transfected with the CB 1 receptor and one of the Gα 12/13 activation sensors, along with the Gα 12 or Gα 13 subunits. Concentration-response curves of HEK293 cells ( top ) or the parental and devoid of G 12/13 (ΔG 12/13 ) HEK293 cells ( bottom ) using the PDZ-RhoGEF-RlucII/rGFP-CAAX ( top and bottom left ) or PKN-RBD-RlucII/rGFP-CAAX ( bottom right ) sensors, pretreated or not with UBO-QIC or PTX ( top ). ( D ) Pharmacological validation of the Gα s activation sensor. HEK293 cells were transfected with the GPBA receptor and the Gα s activation ( left and central ) or the EPAC ( right ) sensors. Left: Concentration-response curves using the Gα s activation sensor in the presence or absence of UBO-QIC or PTX, inhibitors of Gα q or Gα i/o , respectively. Central : Concentration-response activation of the Gα s sensor using CTX, a Gα s activator. Right : Concentration-response curve using the EPAC sensor. Inset ; E max values determined from dose-response curves of inhibitors-pretreated cells. Data are expressed as BRET ratio for the concentration-response curves or expressed in % of respective control cells (E max graphs) and are the mean ± SEM of 3 ( A–C ) or 4 ( D ) independent experiments performed in one replicate. Unpaired t-test ( A–D ): *p < 0.05 and ***p < 0.001 compared to control cells. Figure 2—source data 1. Raw data of .

Article Snippet: Transfected construct ( Homo sapiens ) , FLAG-α 2B AR , Domain Therapeutics North America , N/A , .

Techniques: Biomarker Discovery, Activation Assay, Transfection, Concentration Assay, Control

Journal: eLife

Article Title: Effector membrane translocation biosensors reveal G protein and βarrestin coupling profiles of 100 therapeutically relevant GPCRs

doi: 10.7554/eLife.74101

Figure Lengend Snippet: Concentration-response curves elicited in parental (WT) HEK293 cells or devoid of G s (ΔG s ), G 12/13 (ΔG 12/13 ), G q/11 (ΔG q/11 ), or G i/o (ΔG i/o ) proteins, transfected with the indicated receptor (D 2 , GnRHR, GIP, V 2 , EP 3 , or M 3 ) and one of the Gα i/o , Gα q/11 , or Gα 12/13 activation sensors, along with the indicated Gα subunits. Mock condition corresponded to the response elicited in absence of heterologously expressed Gα subunits (i.e. endogenous G proteins effect). Data are the mean ± SEM of 3 -5 independent experiments performed in one replicate and are expressed as BRET 2 ratio. Data presented in ( I ) are the same as in ( A–B ), but with results expressed as % of maximal response elicited by endogenous G proteins (mock) in WT cells. Figure 2—figure supplement 1—source data 1. Raw date of .

Article Snippet: Transfected construct ( Homo sapiens ) , FLAG-α 2B AR , Domain Therapeutics North America , N/A , .

Techniques: Concentration Assay, Transfection, Activation Assay

Journal: eLife

Article Title: Effector membrane translocation biosensors reveal G protein and βarrestin coupling profiles of 100 therapeutically relevant GPCRs

doi: 10.7554/eLife.74101

Figure Lengend Snippet: HEK293 cells were transfected with the ET A receptor and Gα i/o ( A ), Gα q/11 ( B ), or Gα 12/13 ( C ) activation sensors along with each Gα subunit or control DNA (Mock) as control for response obtained with endogenous Gα proteins. Concentration-response curves in response to endothelin-1 are shown ( left and central ), as well as maximal responses obtained with each Gα subunit. Data are the mean ± SEM of 3 independent experiments performed in one replicate and are expressed as BRET 2 ratio. Unpaired t-test: # p < 0.05, ## p < 0.01 and ### p < 0.001 compared to Mock (without receptor) and one-way ANOVA test: *p < 0.05, **p < 0.01 and ***p < 0.001 compared to Mock + ET A . Figure 2—figure supplement 2—source data 1. Raw date of .

Article Snippet: Transfected construct ( Homo sapiens ) , FLAG-α 2B AR , Domain Therapeutics North America , N/A , .

Techniques: Transfection, Activation Assay, Control, Concentration Assay

Journal: eLife

Article Title: Effector membrane translocation biosensors reveal G protein and βarrestin coupling profiles of 100 therapeutically relevant GPCRs

doi: 10.7554/eLife.74101

Figure Lengend Snippet: ( A ) Concentration-response curves elicited in HEK293 cells transfected with the B 2 receptor and one of the Gα q/11 , Gα i/o , or Gα 12/13 activation sensors, along with increasing quantity of the indicated Gα subunits. Data represent a representative experiment ( B ) Concentration-response curves elicited in HEK293 cells transfected with increasing quantity of the M 3 , D 2 , or AT 1 receptors and the Gα q/11 , Gα i/o , or Gα 12/13 activation sensors, along with the indicated Gα subunits. ( C ) Concentration-response curves elicited in HEK293 cells transfected with the ET A receptor and increasing quantity of effector-RlucII (p63-RhoGEF for Gα q/11 , Rap1GAP for Gα i/o or PDZ-RhoGEF for Gα 12/13 ), along with rGFP-CAAX and the indicated Gα subunits. Data are the mean ± SEM of 3 independent experiments performed in one replicate and are expressed in BRET 2 ratio. Figure 2—figure supplement 3—source data 1. Raw date of .

Article Snippet: Transfected construct ( Homo sapiens ) , FLAG-α 2B AR , Domain Therapeutics North America , N/A , .

Techniques: Concentration Assay, Transfection, Activation Assay

Journal: eLife

Article Title: Effector membrane translocation biosensors reveal G protein and βarrestin coupling profiles of 100 therapeutically relevant GPCRs

doi: 10.7554/eLife.74101

Figure Lengend Snippet: Concentration-response curves elicited in HEK293 cells transfected with the indicated receptor (D 2 , GIP, PTH1, M 3 , ET A , B 1 , FP, or Cys-LT 2 ) and one of the Gα i/o , Gα q/11 , or Gα 12/13 EMTA activation sensors, along with the indicated Gα subunits, or the BRET-based Gαβγ dissociation sensors (Gα-RlucII and GFP10-Gγ 1 for Gα q , Gα 12 , and Gα 13 or GFP10-Gγ 2 for Gα i1 , Gα i2 , and Gα oB , with untagged Gβ 1 ). Data are the mean ± SEM from 3-7 independent experiments performed in one replicate and results are expressed in % of the response obtained for cells treated with vehicle. Figure 2—figure supplement 5—source data 1. Raw date of .

Article Snippet: Transfected construct ( Homo sapiens ) , FLAG-α 2B AR , Domain Therapeutics North America , N/A , .

Techniques: Concentration Assay, Transfection, Activation Assay

Journal: eLife

Article Title: Effector membrane translocation biosensors reveal G protein and βarrestin coupling profiles of 100 therapeutically relevant GPCRs

doi: 10.7554/eLife.74101

Figure Lengend Snippet: Comparison of concentration-response curves elicited by the indicated ligand for a specific pathway, following the stimulation of HEK293 cells expressing endogenous or heterologously expressed receptors. The data presented refer to the ligands where a signal was detected on non-transfected cells (endogenous expression) (See for the curves on light gray and yellow background). Data are the mean ± SEM of at least 3 independent experiments performed in one replicate and expressed in % of the response obtained for cells treated with vehicle. Figure 3—figure supplement 2—source data 1. Raw data of .

Article Snippet: Transfected construct ( Homo sapiens ) , FLAG-α 2B AR , Domain Therapeutics North America , N/A , .

Techniques: Comparison, Concentration Assay, Expressing, Transfection

Journal: eLife

Article Title: Effector membrane translocation biosensors reveal G protein and βarrestin coupling profiles of 100 therapeutically relevant GPCRs

doi: 10.7554/eLife.74101

Figure Lengend Snippet: ( A ) Inverse agonist activity detection. Left : Gα i2 activation in HEK293 cells transfected with the Rap1GAP-RlucII/rGFP-CAAX sensors with untagged Gα i2 and increasing amount of A 1 receptor plasmid. Data are expressed in % of response obtained in control cells (0 ng of A 1 ) and are the mean ± SEM of 4–6 independent experiments performed in two replicates. One Way ANOVA test: ***p < 0.001 compared to control cells. HEK293 cells expressing the Gα i2 activation sensor and control (Mock) or A 1 receptor plasmid were stimulated (10 min) with increasing concentrations of the indicated compound. Data are expressed in % of constitutive response obtained in vehicle-treated A 1 transfected cells and are the mean ± SEM of 4-6 independent experiments performed in one replicate. Right: Gα z activation in HEK293 cells transfected with the Rap1GAP-RlucII/rGFP-CAAX sensors with untagged Gα z and increasing amount of CB 1 receptor plasmid. Data are expressed in % of response obtained in control cells (0 ng of CB 1 ) and are the mean ± SEM of 4 independent experiments performed in one replicate. One Way ANOVA test: ***p < 0.001 compared to control cells. HEK293 cells expressing the Gα z activation sensor and increasing amount of CB 1 receptor plasmid were directly stimulated (10 min) with increasing concentrations of the CB 1 inverse agonist rimonabant. Data are expressed as % of the response obtained in control cells (0 ng of CB 1 ) treated with vehicle and are the mean ± SEM of 4 independent experiments performed in one replicate. ( B ) Ligand-biased detection. Concentration-response curves of AT 1 for the endogenous ligand (Angiotensin II, AngII) and biased agonists [Sar1-Ile4-Ile8] AngII (SII), saralasin or TRV027. G protein and βarrestin2 signaling activity were assessed by EMTA platform. Data are expressed in % of maximal response elicited by AngII and are the mean ± SEM of 3–6 independent experiments performed in one replicate. ( C ) Functional selectivity of naturally occurring receptor variants. Concentration-response curves for WT or E/DRY motif Asp128Asn and Arg129His variants of GPR17 upon agonist stimulation in HEK293 cells co-expressing the indicated EMTA biosensor. Data are expressed in % of maximal response elicited by WT receptor and are the mean ± SEM of 3 independent experiments performed in one replicate. Figure 5—source data 1. Raw data of .

Article Snippet: Transfected construct ( Homo sapiens ) , FLAG-α 2B AR , Domain Therapeutics North America , N/A , .

Techniques: Activity Assay, Activation Assay, Transfection, Plasmid Preparation, Control, Expressing, Concentration Assay, Functional Assay

Journal: eLife

Article Title: Effector membrane translocation biosensors reveal G protein and βarrestin coupling profiles of 100 therapeutically relevant GPCRs

doi: 10.7554/eLife.74101

Figure Lengend Snippet: ( A ) Concentration-response curves of Gα i2 activation elicited by adenosine in HEK293 cells transfected with the Rap1GAP-RlucII/rGFP-CAAX sensors with untagged Gα i2 and A 1 or A 3 receptors. Basal level of G i2 activation detected by the GEMTA sensor in absence of heterologous receptor expression is represented by the interrupted line. Data are expressed as uBRET ratio and are the mean ± SEM of 4 independent experiments performed in one replicate. ( B ) Concentration-response curves of Gα q activation elicited by serotonin in HEK293 cells transfected with the p63-RlucII/rGFP-CAAX sensors with untagged Gα q and increasing amount of 5-HT 2C receptor plasmid. Data are expressed as BRET ratio and are the mean ± SEM of 4 independent experiments performed in one replicate. Figure 5—figure supplement 1—source data 1. Raw data of .

Article Snippet: Transfected construct ( Homo sapiens ) , FLAG-α 2B AR , Domain Therapeutics North America , N/A , .

Techniques: Concentration Assay, Activation Assay, Transfection, Expressing, Plasmid Preparation

Journal: eLife

Article Title: Effector membrane translocation biosensors reveal G protein and βarrestin coupling profiles of 100 therapeutically relevant GPCRs

doi: 10.7554/eLife.74101

Figure Lengend Snippet: HEK293 cells transfected with the Rap1GAP-RlucII/p63-RhoGEF-RlucII/rGFP-CAAX sensors along with Gα z and Gα 15 subunits and the indicated untagged receptor were stimulated with increasing concentrations of the indicated ligand. Data are the mean ± SEM from 3-5 independent experiments performed in one replicate and results are expressed in % of vehicle-treated cells. Figure 6—figure supplement 1—source data 1. Raw data of .

Article Snippet: Transfected construct ( Homo sapiens ) , FLAG-α 2B AR , Domain Therapeutics North America , N/A , .

Techniques: Transfection

Journal: eLife

Article Title: Effector membrane translocation biosensors reveal G protein and βarrestin coupling profiles of 100 therapeutically relevant GPCRs

doi: 10.7554/eLife.74101

Figure Lengend Snippet: ( A ) Test of the G z /G 15 biosensor on a safety target panel. ebBRET signal was measured before and after stimulation with the indicated ligand in HEK293 cells transfected with the combined G z /G 15 biosensor and one of the 24 receptors listed. ( B ) Cross-activation of D 2 and α 2A AR by other natural ligands. For the agonist mode read, HEK293 cells expressing D 2 or α 2A AR and either the Gα i2 , Gα oB , or the βarrestin2 + GRK2 sensors were stimulated with increasing concentrations of the indicated ligand. For the antagonist mode read, cells were pretreated with increasing concentrations of the selective D 2 antagonist eticlopride or the selective α 2A AR antagonist WB4101 before stimulation with an EC 80 of the indicated ligand. Data are the mean ± SEM from 3-4 independent experiments performed in one replicate and expressed in % of the response elicited by dopamine or noradrenaline for D 2 and α 2A AR expressing cells, respectively. ( C ) Indirect ( trans ) activation of CB 1 by acetylcholine. For the agonist mode read, HEK293 cells expressing CB 1 and the Rap1GAP-RlucII/rGFP-CAAX sensors with untagged Gα oB were stimulated with increasing concentrations of the indicated ligand. For the antagonist mode read, same cells were pretreated or not with increasing concentrations of the CB inverse agonist AM-630 ( left ) or the cholinergic antagonist atropine ( central ) before stimulation with an EC 80 of the indicated ligand. To evaluate the contribution of G q/11 -coupled receptor, cells were pretreated with the Gα q inhibitor UBO-QIC and then stimulated with increasing concentrations of the indicated ligand ( right ). Data are the mean ± SEM from 3-5 independent experiments performed in one replicate and expressed in % of the response elicited by WIN55,212–2. Figure 6—source data 1. Raw data of .

Article Snippet: Transfected construct ( Homo sapiens ) , FLAG-α 2B AR , Domain Therapeutics North America , N/A , .

Techniques: Transfection, Activation Assay, Expressing

Journal: eLife

Article Title: Effector membrane translocation biosensors reveal G protein and βarrestin coupling profiles of 100 therapeutically relevant GPCRs

doi: 10.7554/eLife.74101

Figure Lengend Snippet: Concentration-dependent activation of Gα i2 protein by ( A ) endogenous S1P 1 receptor in iPSC-derived cardiomyocytes transfected with heterologous Gα i2 , ( B ) endogenous FPR2 in promyelocytic HL-60 cells transfected with heterologous Gα i2 , ( C ) endogenous FPR2 in promyelocytic HL-60 cells with endogenous G i/o proteins and ( D ) endogenous PAR2 receptor in HEK293 cells with endogenous G i/o proteins. In all cases, cells were co-transfected with the Rap1GAP-RlucII/rGFP-CAAX biosensor. Data are the mean ± SEM of 3-4 independent experiments performed in one replicate and are expressed as BRET 2 ratio in percentage of response induced by vehicle. Figure 7—source data 1. Raw data of .

Article Snippet: Transfected construct ( Homo sapiens ) , FLAG-α 2B AR , Domain Therapeutics North America , N/A , .

Techniques: Concentration Assay, Activation Assay, Derivative Assay, Transfection

Journal: eLife

Article Title: Effector membrane translocation biosensors reveal G protein and βarrestin coupling profiles of 100 therapeutically relevant GPCRs

doi: 10.7554/eLife.74101

Figure Lengend Snippet: G protein expression level detection in HEK293 cells transfected with the Gα i/o , Gα 12/13 , Gα q/11 , or Gα s activation sensors along with the indicated Gα protein or control DNA (Mock). Representative immunoblots of 3 independent experiments are shown. Figure 2—figure supplement 6—source data 1. Original Western blot of .

Article Snippet: Transfected construct ( Homo sapiens ) , FLAG-α 2B AR , Domain Therapeutics North America , N/A , .

Techniques: Expressing, Transfection, Activation Assay, Control, Western Blot

Journal: eLife

Article Title: Effector membrane translocation biosensors reveal G protein and βarrestin coupling profiles of 100 therapeutically relevant GPCRs

doi: 10.7554/eLife.74101

Figure Lengend Snippet:

Article Snippet: Transfected construct ( Homo sapiens ) , FLAG-α 2B AR , Domain Therapeutics North America , N/A , .

Techniques: Functional Assay, Transfection, Construct, Plasmid Preparation, Software, Inverted Microscopy

Journal: Bio-protocol

Article Title: Quantification of The Surface Expression of G Protein-coupled Receptors Using Intact Live-cell Radioligand Binding Assays

doi: 10.21769/bioprotoc.3761

Figure Lengend Snippet: Measurement of the surface expression of GPCRs by intact cell ligand binding assays

Article Snippet: In an Eppendorf tube, add 125 μl of serum-free Opti-MEM and 1 μg of α 2B -AR plasmids, mix gently, and incubate for 5 min at RT.

Techniques: Expressing, Ligand Binding Assay

Journal: eLife

Article Title: Effector membrane translocation biosensors reveal G protein and βarrestin coupling profiles of 100 therapeutically relevant GPCRs

doi: 10.7554/eLife.74101

Figure Lengend Snippet:

Article Snippet: Transfected construct ( Homo sapiens ) , FLAG-α 2B AR , Domain Therapeutics North America , N/A , .

Techniques: Functional Assay, Transfection, Construct, Plasmid Preparation, Software, Inverted Microscopy

Journal: Arthritis Research & Therapy

Article Title: A 2B adenosine receptor activity is reduced in neutrophils from patients with systemic sclerosis

doi: 10.1186/ar1468

Figure Lengend Snippet: Immunoblotting analysis of A 2A and A 2B adenosine receptors (ARs) from systemic sclerosis (SSc) neutrophils and controls. Cells obtained from 26 healthy volunteers and 26 SSc patients were lysed as described in the Methods section. Equal amounts of protein (50 μg) were separated on polyacrylamide gel, blotted and probed with 0.1 μg/ml rabbit anti-human A 2A AR or A 2B AR antibodies. Immunoreactive bands were visualized according to electrogenerated chemiluminescence protocol. A 2A and A 2B AR antibodies recognized immunoreactive bands of 45 kDa and 50 kDa, respectively. (a) Representative experiment performed on neutrophils from one healthy volunteer and one SSc patient. (b) Densitometric analysis of A 2A and A 2B AR immunoreactive bands from 26 healthy volunteers and 26 SSc patients. Graph bars: mean ± standard error of band density, normalized to β-actin. White bars are controls; grey bars are SSc patients.

Article Snippet: A 2A AR and A 2B AR antibodies were supplied by Alpha Diagnostic (San Antonio, TX, USA).

Techniques: Western Blot, Electrochemiluminescence

Journal: Frontiers in Immunology

Article Title: Alpha2B-Adrenergic Receptor Regulates Neutrophil Recruitment in MSU-Induced Peritoneal Inflammation

doi: 10.3389/fimmu.2019.00501

Figure Lengend Snippet: Increased neutrophil recruitment in α 2B AR-Tg mice after MSU stimulation. Wt and α 2B AR-Tg mice were treated with MSU to induce peritonitis. After 6 h, the total PECs were harvested. (A) Quantitative analysis of total number of PECs. (B) The PECs were stained with anti-CD11b, anti-F4/80, and anti-Ly6G antibodies. CD11b + cells were gated for the analysis of macrophage (CD11b + Ly6G − F4/80 + ) and neutrophil (CD11b + Ly6G + F4/80 − ). Representative images were shown. (C) Statistical bar graph showing the percentages of macrophages and neutrophils in total CD11b + cells. (D) The absolute numbers of neutrophils and macrophages in PECs. Data are represented as mean ± SEM ( n = 6–8/group). The results shown are from one of three independent experiments. ** p < 0.01, *** p < 0.001. ns, not significant.

Article Snippet: FVB/NJ mice (wild-type, Wt) and α 2B AR transgenic mice (α 2B AR-Tg) in FVB/NJ background were purchased from the Jackson Laboratory.

Techniques: Staining

Journal: Frontiers in Immunology

Article Title: Alpha2B-Adrenergic Receptor Regulates Neutrophil Recruitment in MSU-Induced Peritoneal Inflammation

doi: 10.3389/fimmu.2019.00501

Figure Lengend Snippet: α 2B AR over-expression does not affect the development of macrophage and neutrophil. The PECs and bone marrow cells were collected from untreated α 2B AR-Tg and Wt mice. (A,B) Percentage of CD11b + F4/80 + macrophages in PECs. The cells were stained with anti-CD11b, anti-F4/80, and anti-Gr1 antibodies. The CD11b+ cells were gated for analysis. The percentage and absolute number of CD11b + F4/80 + macrophages and CD11b + Gr1 + F4/80 − neutrophils in PECs were presented. (C,D) Bone marrow cells were stained with anti-CD11b, anti-F4/80, and anti-Ly6G antibodies. The percentage and absolute number of CD11b + Ly6G + F4/80 − neutrophils in bone marrow cells was shown. The results shown are from one of three independent experiments ( n = 6–8/group).

Article Snippet: FVB/NJ mice (wild-type, Wt) and α 2B AR transgenic mice (α 2B AR-Tg) in FVB/NJ background were purchased from the Jackson Laboratory.

Techniques: Over Expression, Staining

Journal: Frontiers in Immunology

Article Title: Alpha2B-Adrenergic Receptor Regulates Neutrophil Recruitment in MSU-Induced Peritoneal Inflammation

doi: 10.3389/fimmu.2019.00501

Figure Lengend Snippet: α 2B AR overexpression does not affect IL-1β and MIP-2 production in the MSU-induced gout model. Wt and α 2B AR-Tg mice were i.p. injected with MSU to establish the gout model. After 6 h, the peritoneal lavage fluids and PECs were harvested. (A) The IL-1β levels in peritoneal lavage fluid were detected by ELISA, and mRNA expression of PECs was analyzed by real-time PCR. (B) The caspase-1 activity in peritoneal macrophage was measured by flow cytometry analysis. DMSO negative control indicates that no caspase-1 substrates were added. (C,D) The mRNA levels of MIP-2 and α 2B AR in PECs were measured by real-time PCR. The results shown are from one of three independent experiments ( n = 6–8/group). *** p < 0.001. ns, not significant.

Article Snippet: FVB/NJ mice (wild-type, Wt) and α 2B AR transgenic mice (α 2B AR-Tg) in FVB/NJ background were purchased from the Jackson Laboratory.

Techniques: Over Expression, Injection, Enzyme-linked Immunosorbent Assay, Expressing, Real-time Polymerase Chain Reaction, Activity Assay, Flow Cytometry, Negative Control

Journal: Frontiers in Immunology

Article Title: Alpha2B-Adrenergic Receptor Regulates Neutrophil Recruitment in MSU-Induced Peritoneal Inflammation

doi: 10.3389/fimmu.2019.00501

Figure Lengend Snippet: α 2B AR overexpression has no effect on IL-1β and TNFα production by bone marrow derived macrophage. BMMs from α 2B AR-Tg and Wt mice were treated with LPS and/or MSU, with or without the presence of alpha2 agonist ( n = 3/group). Six hours after MSU or LPS challenge, culture supernatants were collected for the detection of IL-1β (A) and TNFα (B) concentrations. Data shown is representative of 3 independent experiments.

Article Snippet: FVB/NJ mice (wild-type, Wt) and α 2B AR transgenic mice (α 2B AR-Tg) in FVB/NJ background were purchased from the Jackson Laboratory.

Techniques: Over Expression, Derivative Assay

Journal: Frontiers in Immunology

Article Title: Alpha2B-Adrenergic Receptor Regulates Neutrophil Recruitment in MSU-Induced Peritoneal Inflammation

doi: 10.3389/fimmu.2019.00501

Figure Lengend Snippet: α 2B AR enhances neutrophil recruitment in MSU-stimulated inflammation. Bone marrow neutrophils from α 2B AR-Tg or Wt mice were stained with V440 and CFSE, respectively. Cells were then mixed at 1:1 ration, followed by i.v. injection into MSU-treated Wt mice (A,B) or α 2B AR-Tg mice (C) . Six hours after MSU challenge, the PECs and spleen were harvested. (A) Representative flow cytometry analysis of neutrophil proportion in the PECs and splenic cells from MSU-treated Wt mice. (B,C) Percentages of neutrophils in PECs and spleen from MSU-treated Wt (B) and α2BAR-Tg mice (C) were analyzed. (D) The peritoneal fluid was collected from Wt and α2BAR-Tg recipients and the absolute number of adoptively transferred neutrophils were analyzed. Data shown are representative of 3 independent experiments ( n = 5/group). * p < 0.05, ** p < 0.01, *** p < 0.001. ns, not significant.

Article Snippet: FVB/NJ mice (wild-type, Wt) and α 2B AR transgenic mice (α 2B AR-Tg) in FVB/NJ background were purchased from the Jackson Laboratory.

Techniques: Staining, Injection, Flow Cytometry

Journal: Frontiers in Immunology

Article Title: Alpha2B-Adrenergic Receptor Regulates Neutrophil Recruitment in MSU-Induced Peritoneal Inflammation

doi: 10.3389/fimmu.2019.00501

Figure Lengend Snippet: α 2B AR overexpression enhances in vitro neutrophils migration. Neutrophils were isolated from α2BAR-Tg and Wt bone marrows and stained with V440 (blue) and CFSE (green), respectively. Labeled cells were mixed at 1:1 ration and used for the Transwell® migration assay in response to MIP2. (A) Upper panel, Representative fluorescent microscopic image showing α2BAR-Tg (Blue) and Wt (Green) neutrophils migrating through the membrane. Lower panel, quantitative analysis showing the migration of α2BAR-Tg and Wt neutrophils. (B) Representative dot plot (upper panel) and quantitative analysis (lower panel) showing the percentages of V440-labeled cells (α2BAR-Tg) and CFSE-labeled cells (Wt) in the bottom well of the Transwell® plate. (C) The Cxcr2 expressions on bone marrows neutrophil with or without treatment of MIP-2 or MIP-2 plus α2BAR agonist clonidine hydrochloride were analyzed by flow cytometry. Data shown are representative of 3 independent experiments. ** p < 0.01, *** p < 0.001.

Article Snippet: FVB/NJ mice (wild-type, Wt) and α 2B AR transgenic mice (α 2B AR-Tg) in FVB/NJ background were purchased from the Jackson Laboratory.

Techniques: Over Expression, In Vitro, Migration, Isolation, Staining, Labeling, Transwell Migration Assay, Membrane, Flow Cytometry