Journal: International Journal of Molecular Sciences

Article Title: P2Y 2 and P2X4 Receptors Mediate Ca 2+ Mobilization in DH82 Canine Macrophage Cells

doi: 10.3390/ijms21228572

Figure Lengend Snippet: Expression of P2X and P2Y receptor mRNA in DH82 cells. RNA was isolated from DH82 cells and cDNA was synthesized and amplified using primer pairs designed to each respective P2RX or P2RY genes, with glyceraldehyde 3-phosphate dehydrogenase ( GAPDH ) as a positive control. Amplification in absence of cDNA was conducted for each primer pair to ensure primer specificity. Amplicons were visualized by agarose gel electrophoresis using GelRed and the GelDoc XR+ imaging system and semi-quantitatively analysed by densitometry. Data shown are the mean ± SEM relative to GAPDH expression from three independent experiments.

Article Snippet: Cells were permeabilized with 0.1% ( w / v ) saponin resuspended in a blocking buffer (2% BSA ( w / v ) in PBS) at room temperature for 15 min and then incubated with anti-P2Y 2 (1:250; Alomone, cat no. APR-102, RRID: AB_2756769) or anti-P2X4 (1:250; Sigma-Aldrich cat no. SAB2500734, RRID:AB_10604119) primary antibody in 2% BSA/PBS at room temperature for 2 h. Cells were washed three times with PBS and then incubated with Alexa Fluor594-conjugated anti-rabbit (1:200; Abcam cat no. ab150080, RRID: AB_2650602), or Alexa Fluor594-conjugated anti-goat (1:200; Abcam cat no. ab150136, RRID: AB_2782994) secondary antibody in 2% BSA/PBS at room temperature for 60 min.

Techniques: Expressing, Isolation, Synthesized, Amplification, Positive Control, Agarose Gel Electrophoresis, Imaging

Journal: International Journal of Molecular Sciences

Article Title: P2Y 2 and P2X4 Receptors Mediate Ca 2+ Mobilization in DH82 Canine Macrophage Cells

doi: 10.3390/ijms21228572

Figure Lengend Snippet: Expression of P2X4 and P2Y 2 receptors in DH82 cells. DH82 cells were fixed, permeabilized and labelled with ( A ) anti-P2X4 or ( B ) anti-P2Y 2 receptor primary antibodies, then with anti-goat 594 or anti-rabbit 594 secondary antibodies, respectively. Cells were imaged by confocal microscopy. Scale bar = 20 µm. Results are representative of three independent experiments.

Article Snippet: Cells were permeabilized with 0.1% ( w / v ) saponin resuspended in a blocking buffer (2% BSA ( w / v ) in PBS) at room temperature for 15 min and then incubated with anti-P2Y 2 (1:250; Alomone, cat no. APR-102, RRID: AB_2756769) or anti-P2X4 (1:250; Sigma-Aldrich cat no. SAB2500734, RRID:AB_10604119) primary antibody in 2% BSA/PBS at room temperature for 2 h. Cells were washed three times with PBS and then incubated with Alexa Fluor594-conjugated anti-rabbit (1:200; Abcam cat no. ab150080, RRID: AB_2650602), or Alexa Fluor594-conjugated anti-goat (1:200; Abcam cat no. ab150136, RRID: AB_2782994) secondary antibody in 2% BSA/PBS at room temperature for 60 min.

Techniques: Expressing, Confocal Microscopy