p2x7 extracellular ecto  (Alomone Labs)

Journal: Antioxidants

Article Title: P2X7 Is Involved in the Mouse Retinal Degeneration via the Coordinated Actions in Different Retinal Cell Types

doi: 10.3390/antiox12010141

Figure Lengend Snippet: P2X7 −/− exerted protection against NaIO 3 -induced photoreceptor injury. ( A ) Representative scotopic ERG responses from WT and P2X7 −/− mice with or without NaIO 3 (25 mg/kg) intraperitoneal injection. Three days after injection, ERG analysis was performed. ( B ) Quantification of the average amplitudes of a and b waves from WT control mice ( n = 8), WT NaIO 3 -treated mice ( n = 8), P2X7 −/− control mice ( n = 8) and P2X7 −/− NaIO 3 -treated mice ( n = 10). ( C ) Quantification of the implicit time of a and b waves from the 4 groups of mice. The data were presented as the mean ± SEM. * p < 0.05 compared with the WT control group, # p < 0.05 compared with the WT control group treated with NaIO 3 .

Article Snippet: Different proteins were determined by immunoblot analysis using antibodies for P2X7 extracellular (ecto) (APR-008 Alomone, Jerusalem, Israel), P2X7 (c-term) (APR-004 Alomone, Jerusalem, Israel), β-actin (sc47778 Santa Cruz, CA, USA) and enhanced Chemiluminescence Reagent Plus (Perkin Elmer, Wellesley, MA, USA).

Techniques: Injection

Journal: Antioxidants

Article Title: P2X7 Is Involved in the Mouse Retinal Degeneration via the Coordinated Actions in Different Retinal Cell Types

doi: 10.3390/antiox12010141

Figure Lengend Snippet: P2X7 knockout protected mice from NaIO 3 -induced retinal epithelium injury. OCT images in posterior pole region ( A ) and optical nerve area ( B ) revealed retinal pigment epithelium degeneration after the mice were treated with 25 mg/kg of NaIO 3 for 3 days. RPE damage (yellow arrow in ( A , B ) was observed in the WT group but not in the P2X7 −/− group treated with NaIO 3 . Hyperreflective spots in both the vitreous region (blue arrow in ( B )) and the retina (orange arrow in ( B ) were shown. The ONL thickness was marked by the red double arrowheads in ( B ). The mice numbers were 8, 8, 8 and 10 for WT control, WT NaIO 3 , P2X7 −/− control and P2X7 −/− NaIO 3 groups, respectively. Scale bar: 200 μm. RPE, retinal pigment epithelium; OS, outer segment; IS, inner segment; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer.

Article Snippet: Different proteins were determined by immunoblot analysis using antibodies for P2X7 extracellular (ecto) (APR-008 Alomone, Jerusalem, Israel), P2X7 (c-term) (APR-004 Alomone, Jerusalem, Israel), β-actin (sc47778 Santa Cruz, CA, USA) and enhanced Chemiluminescence Reagent Plus (Perkin Elmer, Wellesley, MA, USA).

Techniques: Knock-Out

Journal: Antioxidants

Article Title: P2X7 Is Involved in the Mouse Retinal Degeneration via the Coordinated Actions in Different Retinal Cell Types

doi: 10.3390/antiox12010141

Figure Lengend Snippet: Histological H&E examination of retinal injury caused by NaIO 3 was alleviated in P2X7 −/− mice. Retinal morphology and loss of photoreceptors and cell nuclei were evaluated by H&E by light microscopy at 3 days post-intraperitoneal injection of NaIO 3 (25 mg/kg) in WT and P2X7 −/− mice. Shown were representative light microscopic images of H&E staining in retinal sections. Enlargement of cell bodies in the ONL and INL, pigment granules along the RPE layer (black arrows) and ONL thinning were observed. Quantitative analysis of ONL thickness was shown. The mice number was 12 for each group. Scale bar: 200 μm. * p < 0.05, indicating significant decrease in ONL thickness by NaIO 3. # p < 0.05 compared with the WT control group treated with NaIO 3 .

Article Snippet: Different proteins were determined by immunoblot analysis using antibodies for P2X7 extracellular (ecto) (APR-008 Alomone, Jerusalem, Israel), P2X7 (c-term) (APR-004 Alomone, Jerusalem, Israel), β-actin (sc47778 Santa Cruz, CA, USA) and enhanced Chemiluminescence Reagent Plus (Perkin Elmer, Wellesley, MA, USA).

Techniques: Light Microscopy, Injection, Staining

Journal: Antioxidants

Article Title: P2X7 Is Involved in the Mouse Retinal Degeneration via the Coordinated Actions in Different Retinal Cell Types

doi: 10.3390/antiox12010141

Figure Lengend Snippet: Histological examination of PAS and Masson trichrome staining. Representative light microscope images of PAS ( A ) and Masson trichrome ( B ) staining of retinal sections in WT and P2X7 −/− mice at 3 days post-intraperitoneal injection of NaIO 3 (25 mg/kg) were shown. The visible retinal pigment granules were shown as black arrows in both PAS ( A ) and Masson trichrome ( B ) staining. The mice number for PAS staining was 5 for each group and for Masson trichrome staining was 7 for each group. Scale bar: 200 μm.

Article Snippet: Different proteins were determined by immunoblot analysis using antibodies for P2X7 extracellular (ecto) (APR-008 Alomone, Jerusalem, Israel), P2X7 (c-term) (APR-004 Alomone, Jerusalem, Israel), β-actin (sc47778 Santa Cruz, CA, USA) and enhanced Chemiluminescence Reagent Plus (Perkin Elmer, Wellesley, MA, USA).

Techniques: Staining, Light Microscopy, Injection

Journal: Antioxidants

Article Title: P2X7 Is Involved in the Mouse Retinal Degeneration via the Coordinated Actions in Different Retinal Cell Types

doi: 10.3390/antiox12010141

Figure Lengend Snippet: NaIO 3 -induced neuroinflammation in retina was reversed by P2X7 knockout. After 36 h post-injection of NaIO 3 (25 mg/kg), mice retina was collected. ( A ) Total mRNA was extracted and reversely transcribed for quantitative PCR analysis of NLRP3, IL-1β and IL-6 mRNA expression. The mice number for qPCR analysis was 8 for each group. Values were normalized to β-actin gene expression and were expressed relative to the control group. ( B ) Parallelly, serum from mice was collected and subjected to ELISA to determine the amounts of IL-1β, TNF-α and IL-6, respectively. The mice number for ELISA was 5 for each group. The data were presented as the mean ± SEM. * p < 0.05 compared with the WT control group, # p < 0.05 compared with the WT control group treated with NaIO 3 .

Article Snippet: Different proteins were determined by immunoblot analysis using antibodies for P2X7 extracellular (ecto) (APR-008 Alomone, Jerusalem, Israel), P2X7 (c-term) (APR-004 Alomone, Jerusalem, Israel), β-actin (sc47778 Santa Cruz, CA, USA) and enhanced Chemiluminescence Reagent Plus (Perkin Elmer, Wellesley, MA, USA).

Techniques: Knock-Out, Injection, Real-time Polymerase Chain Reaction, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Antioxidants

Article Title: P2X7 Is Involved in the Mouse Retinal Degeneration via the Coordinated Actions in Different Retinal Cell Types

doi: 10.3390/antiox12010141

Figure Lengend Snippet: NaIO 3 -induced caspase expression in retina was reversed by P2X7 knockout . ( A ) After 3 days post-injection of NaIO 3 (25 mg/kg), mice retina was collected, and total mRNA was extracted and reversely transcribed for quantitative PCR analyses of caspase-3, caspase-7 and caspase-8 mRNA expression. The mice number for qPCR analysis was 5 for each group. Values were normalized to β-actin gene expression and were expressed relative to the control group. ( B ) Using the retinal tissues to measure the cellular contents of total NADP, NADPH and NADP/NADPH as per manufacturer’s instructions. The mice number for NADPH activity was 5 for each group. * p < 0.05 compared with the WT control group, # p < 0.05 compared with the WT control group treated with NaIO 3 .

Article Snippet: Different proteins were determined by immunoblot analysis using antibodies for P2X7 extracellular (ecto) (APR-008 Alomone, Jerusalem, Israel), P2X7 (c-term) (APR-004 Alomone, Jerusalem, Israel), β-actin (sc47778 Santa Cruz, CA, USA) and enhanced Chemiluminescence Reagent Plus (Perkin Elmer, Wellesley, MA, USA).

Techniques: Expressing, Knock-Out, Injection, Real-time Polymerase Chain Reaction, Activity Assay

Journal: Antioxidants

Article Title: P2X7 Is Involved in the Mouse Retinal Degeneration via the Coordinated Actions in Different Retinal Cell Types

doi: 10.3390/antiox12010141

Figure Lengend Snippet: Comparison of P2X7 expression and effects of BzATP on cell viability in BV-2, 661W, ARPE-19 and rMC1 cells. ( A ) P2X7 protein level in each cell type was determined by immunoblotting. ( B , C ) Cells were treated with NaIO 3 (30 mM) for the indicated times. P2X7 mRNA level was determined by real-time PCR ( B ) and P2X7 protein was determined by immunoblotting ( C ). ( D ) Each cell type was treated with vehicle or BzATP (200 μM) for 6 or 18 h. Cell death was determined by Annexin V/PI staining with FACS. * p < 0.05, indicating the increased P2X7 gene expression by NaIO 3 ( B ) and the cell death caused by BzATP ( D ).

Article Snippet: Different proteins were determined by immunoblot analysis using antibodies for P2X7 extracellular (ecto) (APR-008 Alomone, Jerusalem, Israel), P2X7 (c-term) (APR-004 Alomone, Jerusalem, Israel), β-actin (sc47778 Santa Cruz, CA, USA) and enhanced Chemiluminescence Reagent Plus (Perkin Elmer, Wellesley, MA, USA).

Techniques: Expressing, Western Blot, Real-time Polymerase Chain Reaction, Staining

Journal: Antioxidants

Article Title: P2X7 Is Involved in the Mouse Retinal Degeneration via the Coordinated Actions in Different Retinal Cell Types

doi: 10.3390/antiox12010141

Figure Lengend Snippet: Schematic representation of P2X7 activation in amplification of NaIO 3 -induced neuroinflammation and retinal cell death. ( Left ) In normal conditions, P2X7 is constitutively and highly expressed in microglia than photoreceptors, RPE and Müller cells. ( Right ) Upon NaIO 3 treatment, P2X7 expression in photoreceptors, Müller cells and RPE cells is increased, and cell death in these cell types, as well as microglia, is evoked. ATP releasing from the dying and damaged cells acts as a DAMP to activate microglia for inflammatory IL-1β and IL-6 gene expression. High concentration of ATP might also promote RPE cell death under NaIO 3 stress.

Article Snippet: Different proteins were determined by immunoblot analysis using antibodies for P2X7 extracellular (ecto) (APR-008 Alomone, Jerusalem, Israel), P2X7 (c-term) (APR-004 Alomone, Jerusalem, Israel), β-actin (sc47778 Santa Cruz, CA, USA) and enhanced Chemiluminescence Reagent Plus (Perkin Elmer, Wellesley, MA, USA).

Techniques: Activation Assay, Amplification, Expressing, Concentration Assay