Journal: Purinergic Signalling

Article Title: N-terminal tagging of human P2X7 receptor disturbs calcium influx and dye uptake

doi: 10.1007/s11302-017-9598-8

Figure Lengend Snippet: a Example of data from pore formation assay showing YO-PRO-1 fluorescence intensity increase over time in a single 96-well with GripTite cells transfected with P2RX7 + empty vector and stimulated with 250 μM BzATP. b Fluorescence intensity baseline for GripTite cells transfected with P2RX7 in combination with empty vector (pcDNA3.1) or a vector for eGFP. There was no statistically significant difference between groups when compared in an unpaired two-sided t test using GraphPad Prism 6. Data are shown as mean + SEM, n = 7–8 from three independent runs

Article Snippet: Spectral overlap testing YO-PRO-1 and Fluo4 have fluorescent spectra that are almost identical to that of eGFP (Semrock SearchLight http://searchlight.semrock.com /).

Techniques: Tube Formation Assay, Fluorescence, Transfection, Plasmid Preparation

Journal: Purinergic Signalling

Article Title: N-terminal tagging of human P2X7 receptor disturbs calcium influx and dye uptake

doi: 10.1007/s11302-017-9598-8

Figure Lengend Snippet: a N-terminal tagging of the human P2X7 receptor disturbs calcium signalling and pore formation. Fluo4 calcium signalling in GripTite cells transiently transfected with different vectors expressing P2RX7 (triangle), C-terminally eGFP-tagged P2RX7 (P2RX7-C-eGFP) (diamond), N-terminally eGFP-tagged P2RX7 (eGFP-N-P2RX7) (circle), and empty vector (cross). Data represents mean ± SEM, n = 4–6 from two independent runs. b YO-PRO-1 dye uptake as a measure of pore formation in GripTite cells transiently transfected with combinations of vector expressing P2RX7 + empty vector (triangle), P2RX7 + eGFP (square), P2RX7-C-eGFP + empty vector (diamond), and eGFP-N-P2RX7 + empty vector (circle). ***p < 0.001, ****p < 0.0001 for P2RX7 + empty vector vs. eGFP-N-P2RX7 + empty vector, #p < 0.05, ###p < 0.001, ####p < 0.0001 for P2RX7 + eGFP vs. eGFP-N-P2RX7 + empty vector, and $p < 0.05 for P2RX7-C-eGFP + empty vector vs. eGFP-N-P2RX7 + empty vector analyzed in two-way ANOVA post hoc Tukey’s multiple comparisons test at respective concentrations using GraphPad Prism 6. Data represents mean ± SEM, n = 3–6 from six independent runs

Article Snippet: Spectral overlap testing YO-PRO-1 and Fluo4 have fluorescent spectra that are almost identical to that of eGFP (Semrock SearchLight http://searchlight.semrock.com /).

Techniques: Transfection, Expressing, Plasmid Preparation

Journal: Purinergic Signalling

Article Title: N-terminal tagging of human P2X7 receptor disturbs calcium influx and dye uptake

doi: 10.1007/s11302-017-9598-8

Figure Lengend Snippet: N-terminal tagging with HA disturbs YO-PRO-1 dye update but not calcium signalling. a Calcium signalling in GripTite cells transiently transfected with different vectors expressing P2RX7 (triangle), N-terminally triple HA-tagged P2RX7 (3HA-N-P2RX7) (half-open triangle), and empty vector (cross). Data represent mean ± SEM, n = 3–4 from two independent runs. b YO-PRO-1 dye uptake as a measure of pore formation in GripTite cells transiently transfected with combinations of a vector expressing P2RX7 (triangle), 3HA-N-P2RX7, or empty vector (cross). Data represent mean ± SEM, n = 3 from three independent runs. c HA immunoreactivity (green) in transiently transfected GripTite cells expressing P2RX7 (left) or 3HA-N-P2RX7 (right). Nuclei were stained with DAPI (blue). Scale = 20 μm

Article Snippet: Spectral overlap testing YO-PRO-1 and Fluo4 have fluorescent spectra that are almost identical to that of eGFP (Semrock SearchLight http://searchlight.semrock.com /).

Techniques: Transfection, Expressing, Plasmid Preparation, Staining