human cannabinoid r2 cb2 cnr2 antibody  (R&D Systems)

Journal: Biomedicines

Article Title: Impact of Δ 9 -Tetrahydrocannabinol on Rheumatoid Arthritis Synovial Fibroblasts Alone and in Co-Culture with Peripheral Blood Mononuclear Cells

doi: 10.3390/biomedicines10051118

Figure Lengend Snippet: Flow cytometric detection of CB 1 and CB 2 in and on RASF. RASF were incubated with TNF (10 ng/mL) for 72 h, and CB 1 and CB 2 levels were determined thereafter. Upper panel: histogram; detection of CB 1 at the plasma membrane (PM) and intracellularly and CB 2 at the plasma membrane. Lower panel: violin plots; quantification of CB 1 and CB 2 . t –test was used for comparisons. p = 0.05 was the level of significance.

Article Snippet: The following antibodies were used: CB 1 (FAB3834R, 0.2 mg/mL, 1:10, R&D Systems/Biotechne, Wiesbaden, Germany), CB 2 (FAB36551G, 0.2 mg/mL, 1:40, R&D Systems/Biotechne), Isotype MsIgG2a-Alexa 488 (IC003G, 5 µL/test, R&D Systems/Biotechne), and Isotype MsIgG2a-Alexa 647 (IC003R, 5 µL/test, R&D Systems/Biotechne5 µL/test); RASF were detached from culture dishes with citrate buffer (135 mM KCl, 15 mM Na 3 C 6 H 5 O 7 ) and centrifuged at 300× g . Cells were resuspended in PBS with 10% FCS and incubated with antibodies for 30 min in the dark at room temperature.

Techniques: Incubation, Membrane

Journal: Biomedicines

Article Title: Impact of Δ 9 -Tetrahydrocannabinol on Rheumatoid Arthritis Synovial Fibroblasts Alone and in Co-Culture with Peripheral Blood Mononuclear Cells

doi: 10.3390/biomedicines10051118

Figure Lengend Snippet: Mean intracellular calcium level changes in RASF in response to THC. ( A ) Intracellular calcium mobilization without TNF pre–stimulation. ( B , C ) Intracellular calcium level regulated by THC (0.1−25 µM) with TNF (10 ng/mL) pre–stimulation for 72 h and extracellular calcium (HBSS; ( B – G )) or without calcium (PBS; ( H – M )). *** p < 0.001, ** p < 0.01, * p < 0.05 for differences between antagonist treatment and control (THC only). ( B , H ) Comparisons of different THC concentrations versus control (no THC). Significant values are given in the graph. ANOVA with Bonferroni post hoc test was used for all comparisons. Rimonanbant, CB 1 inverse agonist; COR170, CB 2 inverse agonist; A967079, TRPA1 antagonist; HC-030031, TRPA1 antagonist; RR = Ruthenium Red, general TRP inhibitor.

Article Snippet: The following antibodies were used: CB 1 (FAB3834R, 0.2 mg/mL, 1:10, R&D Systems/Biotechne, Wiesbaden, Germany), CB 2 (FAB36551G, 0.2 mg/mL, 1:40, R&D Systems/Biotechne), Isotype MsIgG2a-Alexa 488 (IC003G, 5 µL/test, R&D Systems/Biotechne), and Isotype MsIgG2a-Alexa 647 (IC003R, 5 µL/test, R&D Systems/Biotechne5 µL/test); RASF were detached from culture dishes with citrate buffer (135 mM KCl, 15 mM Na 3 C 6 H 5 O 7 ) and centrifuged at 300× g . Cells were resuspended in PBS with 10% FCS and incubated with antibodies for 30 min in the dark at room temperature.

Techniques:

Journal: Biomedicines

Article Title: Impact of Δ 9 -Tetrahydrocannabinol on Rheumatoid Arthritis Synovial Fibroblasts Alone and in Co-Culture with Peripheral Blood Mononuclear Cells

doi: 10.3390/biomedicines10051118

Figure Lengend Snippet: Mean PoPo3 uptake by RASF in response to THC. ( A ) PoPo3 uptake without TNF pre–stimulation, ( B , C ) by THC with TNF (10 ng/mL) pre–stimulation for 72 h and extracellular calcium (HBSS; ( B – I )) or without calcium (PBS; ( J – Q )). *** p < 0.001, ** p < 0.01, * p < 0.05 for differences between antagonist treatment and control (THC only, B,J). ( B , J ) Comparisons of different THC concentrations versus control (no THC). Significant values are given in the graph. ANOVA with Bonferroni post hoc test was used for all comparisons. Rimonanbant, CB 1 inverse agonist; COR170, CB 2 inverse agonist; A967079, TRPA1 antagonist; HC-030031, TRPA1 antagonist; RR = Ruthenium Red, general TRP inhibitor, Decynium-22, organic cation transport inhibitor.

Article Snippet: The following antibodies were used: CB 1 (FAB3834R, 0.2 mg/mL, 1:10, R&D Systems/Biotechne, Wiesbaden, Germany), CB 2 (FAB36551G, 0.2 mg/mL, 1:40, R&D Systems/Biotechne), Isotype MsIgG2a-Alexa 488 (IC003G, 5 µL/test, R&D Systems/Biotechne), and Isotype MsIgG2a-Alexa 647 (IC003R, 5 µL/test, R&D Systems/Biotechne5 µL/test); RASF were detached from culture dishes with citrate buffer (135 mM KCl, 15 mM Na 3 C 6 H 5 O 7 ) and centrifuged at 300× g . Cells were resuspended in PBS with 10% FCS and incubated with antibodies for 30 min in the dark at room temperature.

Techniques:

Journal: Scientific reports

Article Title: Cannabinoid receptor type 2 agonist JWH-133 decreases cathepsin B secretion and neurotoxicity from HIV-infected macrophages.

doi: 10.1038/s41598-021-03896-3

Figure Lengend Snippet: Figure 1. HIV-infected MDM keep expressing surface CB2R at day 12pi. MDM were labeled with a fluorescent CB2R monoclonal antibody (FL4 channel) conjugated with Alexa Fluor® 647 and then analyzed by flow cytometry. (a) Representative histograms of extracellular staining show significant differences in fluorescence intensities from HIV + (dark histogram) and uninfected (grey histogram) on the expression of CB2R compared with the nonfluorescent MDM (unshaded histograms to the left. (b) A graphic representation of surface CB2R levels per donor is shown. Panels a and b are representative of three different donors (n = 3). An unstained control was included as a technical negative control and is representative of 1 donor (n = 1). (c) Representative images of intracellular levels of CB2R and GAPDH expression using Western Blot. Images were cropped from the same blot after stripping and reprobing with a different antibody and were acquired using different exposure times. Full-length blots are presented in Supplementary Fig. 4 (d) A graphic representation of intracellular CB2R levels per donor is shown. Figures c and d are representative of four different donors (n = 4).

Article Snippet: MDM were incubated with an anti-human CB2R-Alexa Fluor® 647 conjugated antibody (R&D Systems) (1:100 in PBS 1X) for 1 h at 4 °C.

Techniques: Infection, Expressing, Labeling, Flow Cytometry, Staining, Fluorescence, Control, Negative Control, Western Blot, Stripping Membranes

Journal: Scientific reports

Article Title: Cannabinoid receptor type 2 agonist JWH-133 decreases cathepsin B secretion and neurotoxicity from HIV-infected macrophages.

doi: 10.1038/s41598-021-03896-3

Figure Lengend Snippet: Figure 2. CB2R agonists treatment decrease HIV-1 replication and CATB secretion from MDM. MDM were infected with HIV-1 and treated with CB2R agonists, JWH-133 and HU-308. HIV-1 p24 and CATB levels were measured from supernatants of days 3, 6, 9, and 12pi by ELISA. (a) Temporal HIV-1 p24 levels in supernatants of HIV-infected MDM treated with JWH-133. (b) Temporal CATB secretion levels in supernatants of HIV- infected MDM treated with JWH-133. Figures a and b are representative of at least four different donors (n = at least 4). (c) Temporal HIV-1 p24 levels in supernatants of HIV-infected MDM treated with HU-308. (d) Temporal CATB secretion levels in supernatants of HIV-infected MDM treated with HU-308. Figures c and d are representative of at least six different donors (n = at least 6). Graphs are presented using the mean and the ± standard error of the mean (SEM). *p < 0.05, **p < 0.01 vs. vehicle control at its respective time-point.

Article Snippet: MDM were incubated with an anti-human CB2R-Alexa Fluor® 647 conjugated antibody (R&D Systems) (1:100 in PBS 1X) for 1 h at 4 °C.

Techniques: Infection, Enzyme-linked Immunosorbent Assay, Control

Journal: Scientific reports

Article Title: Cannabinoid receptor type 2 agonist JWH-133 decreases cathepsin B secretion and neurotoxicity from HIV-infected macrophages.

doi: 10.1038/s41598-021-03896-3

Figure Lengend Snippet: Figure 3. JWH-133 prevents HIV-induced increase in surface CB2R expression and induces oscillating expressions over time. MDM were cultured in 8-well chamber slides, infected with HIV-1ADA, and treated with JWH-133 at 0.5 µM. Slides were fixed at days 3, 6, 9, and 12dpi. Cells were stained with an anti-CB2R antibody (red) and nuclei were stained with DAPI (blue). At least 3 different random pictures per condition were acquired. Representative immunofluorescence images are shown. Graphs are presented using the mean ± standard error of the mean (SEM). This figure is representative of three different donors (n = 3). *p < 0.05, **p < 0.01.

Article Snippet: MDM were incubated with an anti-human CB2R-Alexa Fluor® 647 conjugated antibody (R&D Systems) (1:100 in PBS 1X) for 1 h at 4 °C.

Techniques: Expressing, Cell Culture, Infection, Staining, Immunofluorescence

Journal: Scientific reports

Article Title: Cannabinoid receptor type 2 agonist JWH-133 decreases cathepsin B secretion and neurotoxicity from HIV-infected macrophages.

doi: 10.1038/s41598-021-03896-3

Figure Lengend Snippet: Figure 4. JWH-133 decreases HIV-1 replication and CATB secretion through CB2R activation. MDM were cultured in 24-wells plates and infected with HIV-1ADA. After removal of residual virus, cells were treated with CB2R antagonist SR144528 (SR: 1 µM) for 1 h, followed by JWH-133 (JWH) treatment at 0.5 µM. Treatments were maintained for 6dpi, exchanging half of the media at day 3pi and repeating the co-administration protocol. HIV-1 p24 and CATB levels were measured in HIV-infected MDM supernatants using ELISA. HIV-1 p24 and CATB levels were normalized against its vehicle control per donor. (a) HIV-1 p24 levels in HIV-infected MDM after co-administration of CB2R ligands. This figure is representative of at least eight different donors (n = 8). (b) CATB levels in HIV-infected MDM after co-administration of CB2R ligands. This figure is representative of ten different donors (n = 10). Graphs are presented using the mean ± standard error of the mean (SEM). *p < 0.05, **p < 0.01.

Article Snippet: MDM were incubated with an anti-human CB2R-Alexa Fluor® 647 conjugated antibody (R&D Systems) (1:100 in PBS 1X) for 1 h at 4 °C.

Techniques: Activation Assay, Cell Culture, Infection, Virus, Enzyme-linked Immunosorbent Assay, Control