Journal: Cancer Cell International

Article Title: The inhibitory receptors PD1, Tim3, and A2aR are highly expressed during mesoCAR T cell manufacturing in advanced human epithelial ovarian cancer

doi: 10.1186/s12935-023-02948-0

Figure Lengend Snippet: Baseline expression level of inhibitory receptors in T cells of patients with EOC and healthy controls. The baseline percentage of TIM3+ (A) , PD1+ (B) , and A2aR+ (C) cells are significantly higher in EOC patient-derived T cells post thawing. The frequency of T tex cells is significantly higher in patients’ samples compared to normal controls (D) . However, the frequency of T pex cells is not significant different between patients and healthy samples (D). Representative overlay histogram plots representing expression level of TIM3 (A), PD1 (B), and A2aR (C) in EOC patient-derived T cells sample (Red color) vs. unstained sample (Blue color). Representative overlay dot plot (D) representing the frequency of terminally exhausted and progenitor exhausted T cells in an EOC patient sample (Red color) vs. unstained sample (Blue color). Data from healthy donors (n = 30) and EOC patients (n = 30) were analyzed using Welch’s t-test (A and B) Mann-Whitney U test (C and D). * P < 0.05; **P < 0.01; ***P < 0.001. Data are presented as mean ± SD.

Article Snippet: Alexa Fluor® 647 anti-human Adenosine A2aR (Cat.no: FAB94971R, clone #599,717), and isotype control (Cat.no: IC003R, clone #20,102) were purchased from R&D Systems.

Techniques: Expressing, Derivative Assay, MANN-WHITNEY

Journal: Cancer Cell International

Article Title: The inhibitory receptors PD1, Tim3, and A2aR are highly expressed during mesoCAR T cell manufacturing in advanced human epithelial ovarian cancer

doi: 10.1186/s12935-023-02948-0

Figure Lengend Snippet: Expression pattern of inhibitory receptors during CAR T manufacturing process. CAR T cell population positive for TIM3 (A) , PD1 (B) , and A2aR (C) in EOC patient-derived T cells during CAR T cell manufacturing steps. The frequency of T pex (D) and T tex (E) cells during manufacturing steps of CAR T cells derived from EOC patient T cells. Median fluorescence intensity (MFI) of A2aR (F) , TIM3 (G) , and PD1 (H) in EOC patient-derived T cells during CAR T cells manufacturing process. Healthy donors (n = 30) and EOC patients (n = 30) data were analyzed using RM one-way ANOVA and Tukey multiple comparison test. * P < 0.05; **P < 0.01; ***P < 0.001. Data are presented as mean ± SD.

Article Snippet: Alexa Fluor® 647 anti-human Adenosine A2aR (Cat.no: FAB94971R, clone #599,717), and isotype control (Cat.no: IC003R, clone #20,102) were purchased from R&D Systems.

Techniques: Expressing, Derivative Assay, Fluorescence, Comparison

Journal: Cancer Cell International

Article Title: The inhibitory receptors PD1, Tim3, and A2aR are highly expressed during mesoCAR T cell manufacturing in advanced human epithelial ovarian cancer

doi: 10.1186/s12935-023-02948-0

Figure Lengend Snippet: Expression pattern of inhibitory receptors in patient-derived CAR T cells. CAR T cell population positive for TIM3 (A) , PD1 (B) , and A2aR (C) in EOC patient-derived T cells after activation and transduction vs. healthy controls. The frequency of T tex (D) and T pex (E) cells after activation and transduction of EOC patient-derived T cells vs. healthy controls. Data from healthy donors (n = 30) and EOC patients (n = 30) were analyzed using Mann-Whitney U test (A, B, and D) and Welch’s t-test (C and E). * P < 0.05; **P < 0.01; ***P < 0.001. Data are represented by mean ± SD.

Article Snippet: Alexa Fluor® 647 anti-human Adenosine A2aR (Cat.no: FAB94971R, clone #599,717), and isotype control (Cat.no: IC003R, clone #20,102) were purchased from R&D Systems.

Techniques: Expressing, Derivative Assay, Activation Assay, Transduction, MANN-WHITNEY

Journal: Cancer Cell International

Article Title: The inhibitory receptors PD1, Tim3, and A2aR are highly expressed during mesoCAR T cell manufacturing in advanced human epithelial ovarian cancer

doi: 10.1186/s12935-023-02948-0

Figure Lengend Snippet: Differential expression of immune inhibitory receptors in different cancer stages during CAR T cell manufacturing. Cell surface expression levels of TIM3 (A) , PD1 (B) , A2aR (C) , and the frequency of T pex (D) and T tex (E) cells at different steps of CAR T cell manufacturing process for different stages of EOC. Data from stage III patients (n = 18) and stage IV patients (n = 12) were analyzed using Welch’s t-test. * P < 0.05; **P < 0.01; ***P < 0.001. Data are presented by mean ± SD.

Article Snippet: Alexa Fluor® 647 anti-human Adenosine A2aR (Cat.no: FAB94971R, clone #599,717), and isotype control (Cat.no: IC003R, clone #20,102) were purchased from R&D Systems.

Techniques: Quantitative Proteomics, Expressing

Journal: Cancer Cell International

Article Title: The inhibitory receptors PD1, Tim3, and A2aR are highly expressed during mesoCAR T cell manufacturing in advanced human epithelial ovarian cancer

doi: 10.1186/s12935-023-02948-0

Figure Lengend Snippet: Effect of CarboTaxol treatment on T cell expansion, frequency, and expression pattern of immune inhibitory receptors during CAR T cell manufacturing process. Fold increase of T cells from CarboTaxol-treated and untreated patients post T cell activation (A) . CD3 expression after T cell activation in CarboTaxol treated and newly diagnosed patients (B) . The percentage of A2aR (C) , TIM3 (D) , PD1 (E) positive cells, and the frequency of T tex (F) and T pex cells (G) during CAR T cell manufacturing process in CarboTaxol-treated and newly diagnosed patients. Data from CarboTaxol-treated patients (n = 20) and new patients (n = 10) were analyzed using Welch’s t-test. * P < 0.05; **P < 0.01; ***P < 0.001. Data are presented by mean ± SD.

Article Snippet: Alexa Fluor® 647 anti-human Adenosine A2aR (Cat.no: FAB94971R, clone #599,717), and isotype control (Cat.no: IC003R, clone #20,102) were purchased from R&D Systems.

Techniques: Expressing, Activation Assay

Journal: Journal of Immunology (Baltimore, Md. : 1950)

Article Title: Involvement of Adenosine A 2A Receptors in Engulfment-Dependent Apoptotic Cell Suppression of Inflammation 1

doi: 10.4049/jimmunol.1002284

Figure Lengend Snippet: (A) Wild-type peritoneal macrophages were exposed for one hr. to apoptotic thymocytes alone or (B) in combination with 0.1 μg/ml cycloheximide or 10 μM actinomycin D. Thymocytes were then washed away and macrophages were further incubated (A) for the indicated time periods or for (B) 3 hrs. Cell surface expression of A2A receptor was determined by flow cytometry. (C–D) Blocking of apoptotic cell phagocytosis abolishes the increase of A2A receptor level. Phagocytosis was inhibited by pretreating macrophages with 50 mM cytochalasin D or by masking the phosphatidylserine on the apoptotic cell surface with recombinant annexin V (10 μg / 105 apoptotic cell). After one hr. co-incubation apoptotic cell were washed away and macrophages were further cultured for 2hrs (C) or 3 hrs (D). Adenosine A2A receptor level was determined by quantitative PCR (C) or by flow cytometry (D). Macrophages were treated with GW501516, a PPARδ agonist (E), or by 22 (R)OH-cholesterol, an LXR agonist (F), for 3 hrs. Cell surface expression of A2A receptor was determined by flow cytometry. A2AR gene expression level was also determined in PPARδ knock bone marrow derived (G) and LXR null peritoneal macrophages (H). Macrophages were coincubated with apoptotic thymocytes for 1 hr. Apoptotic cells were then washed away and macrophages were further incubated for 2 hrs. The gene expression levels were measured by quantitative PCR. MFI: mean fluorescence intensity. Results are expressed as mean ± S.D. of three or four independent experiments (*p<0.05).

Article Snippet: After the treatments macrophages were washed (1× PBS), collected, blocked with 50% FBS for 30 min. and labeled with anti-mouse A2AR antibody (BD Pharmingen) or goat IgG isotype control.

Techniques: Incubation, Expressing, Flow Cytometry, Blocking Assay, Recombinant, Cell Culture, Real-time Polymerase Chain Reaction, Derivative Assay, Fluorescence

Journal: Journal of Immunology (Baltimore, Md. : 1950)

Article Title: Involvement of Adenosine A 2A Receptors in Engulfment-Dependent Apoptotic Cell Suppression of Inflammation 1

doi: 10.4049/jimmunol.1002284

Figure Lengend Snippet: (A) The map of the 40 cytokines detected on the membranes. (B) Cytokine panel of control and apoptotic cell treated wild-type and adenosine A2A receptor null peritoneal macrophages. Peritoneal macrophages were co-incubated with apoptotic thymocytes for one hr. (MPh:APO=1:10) followed by removal of apoptotic cells and addition of fresh medium. Supernatants were collected five hrs. later and cytokine levels were determined by cytokine array. Arrows highlight MIP-2, which is significantly overproduced by A2A null macrophages. (C) Cytokines, which levels were significantly different (p<0.05) in the supernatants analyzed by cytokine array. (D) Flow cytometric analysis of AnnexinV-FITC and propidium iodide stained apoptotic thymocytes. (E) Flow cytometric measurement of capacity of wild type and A2A null peritoneal macrophages to take up apoptotic thymocytes. (F–G) The number of engulfed fluorescently labeled apoptotic cells within WT or A2AR-null macrophages counted by confocal microscopy following 1h of phagocytosis. “s.b.” indicates surface bound cells. Results are expressed as mean ± S.D. of three independent experiments.

Article Snippet: After the treatments macrophages were washed (1× PBS), collected, blocked with 50% FBS for 30 min. and labeled with anti-mouse A2AR antibody (BD Pharmingen) or goat IgG isotype control.

Techniques: Incubation, Staining, Labeling, Confocal Microscopy

Journal: Journal of Immunology (Baltimore, Md. : 1950)

Article Title: Involvement of Adenosine A 2A Receptors in Engulfment-Dependent Apoptotic Cell Suppression of Inflammation 1

doi: 10.4049/jimmunol.1002284

Figure Lengend Snippet: (A) Cytokine panel of control, apoptotic cell exposed and SCH442416 (A2AR-specific antagonist) treated wild-type macrophages. Peritoneal macrophages were pre-incubated with 10 nM SCH442416 for 30 min, then they were exposed to apoptotic thymocytes for one hr (MPh:APO=1:10). 5 hours after the removal of apoptotic cells supernatants were collected and cytokine levels were determined by cytokine array. Arrows highlight KC (B), MIP-2 (C), MIP-1α (D) and β (E) overproduced by SCH442416 treated macrophages. (F) MIP-2, (G) KC and (H) active TGF-β production of wild type and A2AR null macrophages engulfing apoptotic cells determined by ELISA at the same time point as above. Results are expressed as mean ± S.D. of three independent experiments (*p<0.05).

Article Snippet: After the treatments macrophages were washed (1× PBS), collected, blocked with 50% FBS for 30 min. and labeled with anti-mouse A2AR antibody (BD Pharmingen) or goat IgG isotype control.

Techniques: Incubation, Enzyme-linked Immunosorbent Assay

Journal: Journal of Immunology (Baltimore, Md. : 1950)

Article Title: Involvement of Adenosine A 2A Receptors in Engulfment-Dependent Apoptotic Cell Suppression of Inflammation 1

doi: 10.4049/jimmunol.1002284

Figure Lengend Snippet: (A) Wild-type and A2A receptor null mice with sterile peritonitis were intraperitoneally injected with apoptotic cells (105 cells / mouse in physiological saline (apo. cell) or vehicle alone. Untreated control mice were uninjected. In some cases neutralizing MIP-2 and neutralizing KC antibodies (nMIP-2 and nKC abs) or their isotype controls were also injected. Three hrs. later peritoneal cells were collected and analyzed by flow cytometry for Gr-1-positivity. The levels of MIP-2 (B) and KC (C) in the lavage fluids were determined by ELISA at the same time point. Results are expressed as mean ± S.D. of five independent experiments (*p<0.05).

Article Snippet: After the treatments macrophages were washed (1× PBS), collected, blocked with 50% FBS for 30 min. and labeled with anti-mouse A2AR antibody (BD Pharmingen) or goat IgG isotype control.

Techniques: Injection, Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: Journal of Immunology (Baltimore, Md. : 1950)

Article Title: Involvement of Adenosine A 2A Receptors in Engulfment-Dependent Apoptotic Cell Suppression of Inflammation 1

doi: 10.4049/jimmunol.1002284

Figure Lengend Snippet: (A) Adenosine A2A receptor null peritoneal macrophages were exposed to apoptotic cells alone or after a 30 min. pretreatment with forskolin (10 μM), cholera toxin (100 ng / ml) or dibutyryl-cAMP (100 μM). (B) Wild-type peritoneal macrophages were exposed to apoptotic cells alone or in combination with 100 μM Rp-cAMP triethylamine or adenosine deaminase (1 U/ml). (C) Wild type or adenosine A2A receptor null peritoneal macrophages were exposed to apoptotic cells alone or after a 30 min. pretreatment with 5 μg / ml actinomycin D or 0.1 μg / ml cycloheximide. In all these experiments (A–C) apoptotic cells were washed away after one hr. and supernatants were collected after 5 hrs. to determine MIP-2 production by ELISA. (D) Quantitative RT-PCR analysis of MIP-2 mRNA expression in wild-type resting, one hr. apoptotic cell exposed peritoneal macrophages at 2 hours after the removal of the apoptotic cells or apoptotic thymocytes. Expression values are represented as mean amount of target mRNA normalized to the expression of cyclophilin. Results are expressed as mean ± S.D. of five independent experiments (*p<0.05).

Article Snippet: After the treatments macrophages were washed (1× PBS), collected, blocked with 50% FBS for 30 min. and labeled with anti-mouse A2AR antibody (BD Pharmingen) or goat IgG isotype control.

Techniques: Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Expressing

Journal: Journal of Immunology (Baltimore, Md. : 1950)

Article Title: Involvement of Adenosine A 2A Receptors in Engulfment-Dependent Apoptotic Cell Suppression of Inflammation 1

doi: 10.4049/jimmunol.1002284

Figure Lengend Snippet: L-NAME, a nitric oxide synthetase inhibitor, prevents apoptotic cell-induced MIP-2 production by macrophages on both protein (A) and (B) mRNA level in a dose dependent manner. Wild type or adenosine A2A receptor null peritoneal macrophages were exposed to apoptotic cells for one hr. alone or after a 30 min. pretreatment with L-NAME in the indicated concentrations. After one hr. apoptotic cells were washed away and mRNA levels were determined 2 hrs later, while cytokine levels in the supernatant 5 hrs. later, L-NAME being constantly present. mRNA levels are expressed as fold changes as compared to the resting macrophages. (C) NO production of macrophages exposed to apoptotic cells is enhanced in the absence of A2AR. Macrophages were exposed to apoptotic cells for one hr. NO production was determined following an additional hr. (D) The NO donor sodium nitropusside enhances MIP-2 production, but only in macrophages exposed to apoptotic cells. Wild type peritoneal macrophages were exposed to the indicated concentrations of sodium nitropusside in the presence or absence of apoptotic cells. Apoptotic cells were washed away after one hr. MIP-2 mRNA levels were determined 2 hrs. later. (E) Forskolin, an adenylate cyclase activator, decreases NO production by A2AR−/− macrophages. A2AR−/− peritoneal macrophages were exposed to apoptotic cells for one hr. alone or after a 30 min. pretreatment with forskolin (10 μM). Apoptotic cells were washed away and NO production was determined following an additional hr. (F) Inhibition of protein kinase A enhances NO production of wild-type macrophages exposed to apoptotic cells. Wild-type peritoneal macrophages were exposed to apoptotic cells for one hr. alone or after a 30 min. pretreatment with 100μM Rp-cAMP triethylamine. Apoptotic cells were washed away and NO production was determined following an additional hr. Results are expressed as mean ± S.D. of five independent experiments (*p<0.05).

Article Snippet: After the treatments macrophages were washed (1× PBS), collected, blocked with 50% FBS for 30 min. and labeled with anti-mouse A2AR antibody (BD Pharmingen) or goat IgG isotype control.

Techniques: Inhibition

Journal: Journal of Immunology (Baltimore, Md. : 1950)

Article Title: Involvement of Adenosine A 2A Receptors in Engulfment-Dependent Apoptotic Cell Suppression of Inflammation 1

doi: 10.4049/jimmunol.1002284

Figure Lengend Snippet: (A) mRNA expression of arginase I and II and iNOS in resting wild type and A2AR−/− macrophages. Expression values are represented as mean amount of target mRNA normalized to the expression of cyclophilin. (B) Changes in the mRNA expression of arginase I and II and iNOS in wild type and A2AR−/− macrophages following exposure to apoptotic cells. mRNA levels are expressed as fold changes as compared to the resting macrophages. (C) Inhibition of protein kinase A in wild-type macrophages enhances, while (D) activation of adenylate cyclase in adenosine A2A receptor null macrophages decreases the mRNA levels of iNOS. The expression of arginase II (lined bars) under the same conditions is adversely affected. Results are expressed as mean ± S.D. of five independent experiments (*p<0.05).

Article Snippet: After the treatments macrophages were washed (1× PBS), collected, blocked with 50% FBS for 30 min. and labeled with anti-mouse A2AR antibody (BD Pharmingen) or goat IgG isotype control.

Techniques: Expressing, Inhibition, Activation Assay

Journal: Cell reports. Medicine

Article Title: Adenosine A2A receptor is a tumor suppressor of NASH-associated hepatocellular carcinoma.

doi: 10.1016/j.xcrm.2023.101188

Figure Lengend Snippet: Figure 1. Development of spontaneous and carcinogen-induced HCC is enhanced in A2AR-deficient mice (A) Experimental setting: WT and A2AR-deficient males were aged to 80 weeks old. Representative pictures of spontaneous HCC observed in WT and A2AR- deficient males are shown. Scale bar represents 2 cm. (B) HCC incidence (observation of macroscopic lesions) in 80-week-old WT and A2AKO males. (C) Hematoxylin and eosin staining of cancerous livers collected from A2AKO mice depicting the different stages of HCC: top panel, stages 1 and 2, and bottom panel, stages 3 and 4; scale bar represents 50 mm. An asterisk (*) indicates trabeculae that are present in stage II–IV mouse liver tumors. (D) Experimental setting: WT and A2AR-deficient pups were treated with DMBA, 3 to 4 days after birth, and aged until 40 weeks old.

Article Snippet: Protein block solution (Dako, 100%) was added for 30 min prior to an overnight incubation at 4 C with primary antibodies: anti-human/mouse A2AR mAb (clone 7F6-G5-A2, Novus biologicals) and efluor 550-coupled anti-F4/80 mAb (clone BM8, Biolegend).

Techniques: Staining

Journal: Cell reports. Medicine

Article Title: Adenosine A2A receptor is a tumor suppressor of NASH-associated hepatocellular carcinoma.

doi: 10.1016/j.xcrm.2023.101188

Figure Lengend Snippet: Figure 2. Loss of A2AR signaling triggers spontaneous obesity, NASH, and systemic inflammation (A) Weight gain of WT, A2A/, and A2A+/ mice (males) was monitored weekly starting from week 4 to 32 weeks old. (B) Body composition of 24-week-old WT and A2AKO males. (C) Representative picture of 32-week-old WT and A2AKO mice showing fat accumulation in A2AKO animals.

Article Snippet: Protein block solution (Dako, 100%) was added for 30 min prior to an overnight incubation at 4 C with primary antibodies: anti-human/mouse A2AR mAb (clone 7F6-G5-A2, Novus biologicals) and efluor 550-coupled anti-F4/80 mAb (clone BM8, Biolegend).

Techniques:

Journal: Cell reports. Medicine

Article Title: Adenosine A2A receptor is a tumor suppressor of NASH-associated hepatocellular carcinoma.

doi: 10.1016/j.xcrm.2023.101188

Figure Lengend Snippet: Figure 3. Myeloid- and hepatocyte-specific deletions of A2AR signaling promotes DMBA-induced HCC development (A) Experimental setting: lyzM-Cre+/ A2ARflox/flox

Article Snippet: Protein block solution (Dako, 100%) was added for 30 min prior to an overnight incubation at 4 C with primary antibodies: anti-human/mouse A2AR mAb (clone 7F6-G5-A2, Novus biologicals) and efluor 550-coupled anti-F4/80 mAb (clone BM8, Biolegend).

Techniques:

Journal: Cell reports. Medicine

Article Title: Adenosine A2A receptor is a tumor suppressor of NASH-associated hepatocellular carcinoma.

doi: 10.1016/j.xcrm.2023.101188

Figure Lengend Snippet: Figure 4. Exacerbated development of HCC in A2AR-deficient mice is TNF-a, IL-1b, and IL-17A dependent (A) Experimental setting: WT and A2AKO pups were treated with DMBA. At week 20, A2AKO mice were split into 5 groups receiving isotype control antibody or neutralizing monoclonal antibody (mAb) against TNF-a, IL-1b, IL-17A, and IL-6. Mice were sacrificed at 40 weeks old to evaluate tumor burden. (B) Representative pictures showing livers of 40-week-old A2AR-deficient mice treated with isotype control or anti-cytokine neutralizing mAbs. (C) Mice weights at 40 weeks old. (D) Total liver tumor burden. (E) Tumor incidence. (F) Number of macroscopic nodules. Results are representative of 1 biological replicate, and sample size is indicated for each panel. Data are presented as means ± SEM. Statistical tests: multiple t tests with Benjamin-Hochberg multiple comparison correction (C), Kruskal-Wallis with Dunn’s correction (D and F), or chi-squared test (E).

Article Snippet: Protein block solution (Dako, 100%) was added for 30 min prior to an overnight incubation at 4 C with primary antibodies: anti-human/mouse A2AR mAb (clone 7F6-G5-A2, Novus biologicals) and efluor 550-coupled anti-F4/80 mAb (clone BM8, Biolegend).

Techniques: Control, Comparison

Journal: Cell reports. Medicine

Article Title: Adenosine A2A receptor is a tumor suppressor of NASH-associated hepatocellular carcinoma.

doi: 10.1016/j.xcrm.2023.101188

Figure Lengend Snippet: Figure 5. Pharmacological blockade of A2AR signaling promotes HFD-induced obesity and myeloid-derived liver inflammation (A) Growth curve. WT male mice were fed with a CD-HFD and treated daily with KW6002 (10 mg/kg p.o.), CGS21680 (0.5 mg/kg i.p.), or vehicle for a total of 8 weeks. Mice weight was monitored every week. (B) After 8 weeks of treatment, fat mass was measured using an EchoMRI. (C) Growth curve of male C57BL/6 mice fed with a CD-HFD and treated daily with NIR178 (10 mg/kg p.o.) or vehicle. (D) Fat mass measurement after 8 weeks of treatment with NIR178 using an EchoMRI.

Article Snippet: Protein block solution (Dako, 100%) was added for 30 min prior to an overnight incubation at 4 C with primary antibodies: anti-human/mouse A2AR mAb (clone 7F6-G5-A2, Novus biologicals) and efluor 550-coupled anti-F4/80 mAb (clone BM8, Biolegend).

Techniques: Derivative Assay, Mass Measurement

Journal: Cell reports. Medicine

Article Title: Adenosine A2A receptor is a tumor suppressor of NASH-associated hepatocellular carcinoma.

doi: 10.1016/j.xcrm.2023.101188

Figure Lengend Snippet: Figure 6. Diet-induced steatohepatitis im- pairs the anti-HCC activity of A2AR inhibi- tion (A) Experimental design of the hydrodynamic tail- vein HCC model. (B) Representative pictures of livers collected on mice treated with KW6002 or vehicle subjected to regular chow diet or CD-HFD feeding. (C) Tumor incidence as determined by visualization of macroscopic nodule on livers. (D) Total tumor burden on livers. (E) Liver-to-body weight ratio as a surrogate of total tumor burden. (F) Experimental design: male WT or A2AKO animals were exposed to DEN (50 mg/kg i.p.) at 3 weeks old and fed with a CD-HFD until sacrifice. From weeks 16 to 32, mice were treated daily with KW6002 (10 mg/kg p.o.) or vehicle as indicated. (G) Growth curve of the different group of mice. (H) Total liver tumor burden at sacrifice. (I) Number and size of macroscopic HCC nodules. Results are representative of 1 biological replicate, and sample size is indicated for each panel. Data are presented as means ± SEM. Statistical tests: chi-squared test (C), Mann-Whitney (D, E, H, and I), or Student’s t tests (D).

Article Snippet: Protein block solution (Dako, 100%) was added for 30 min prior to an overnight incubation at 4 C with primary antibodies: anti-human/mouse A2AR mAb (clone 7F6-G5-A2, Novus biologicals) and efluor 550-coupled anti-F4/80 mAb (clone BM8, Biolegend).

Techniques: Activity Assay, MANN-WHITNEY