Modulation of migration by nucleotides in a pleurisy model.

<p>Rats were sensitized with OVA in the first day. On day 14 all animals were treated or not with agonists and antagonists of P2R, and after 1h animals were of challenged. After 24h, cells in the pleural cavity was collected and counted for total cells (Fig 5A) and eosinophils (Fig 5B). *** p<0. 05 when saline group was compared to other treatments; +++ p<0.01 when UTP group was compared to ATPγS group; ++ p<0.05 when UTP group was compared to ATP group; # p<0.05 when ATP group was compared to Suramin group; ### p<0.01 when UTP group was compared to Suramin group.This experiment was realized for 3 times with 6 animals per group.</p

Analysis of calcium transients activated by selective P2Y nucleotides.

<p>Rat eosinophils were incubated at 37°C. 2 μM ionomycin was used as positive control. A- 500 μM ATP, 500 μM UTP, 1 μM MRS 4062 and 1 μM 2-S-UTP application on mouse peritoneal macrophages at 37°C for 3 minutes. All recordings were performed at distinct wells. B- Bar graph representation of 2–3 independent experiments. C- 500 μM ATP, 500 μM UTP, 1 μM MRS 4062 and 1 μM 2-S-UTP application on rat peritoneal macrophages at 37°C for 3 minutes. All recordings were performed at distinct wells. D- Bar graph representation of 2–3 independent experiments. *** p<0.01 or * p<0.05 when baseline (saline) was compared to other treatment.</p

Analysis of calcium transients activated by nucleotides.

<p>Rat eosinophils were incubated at 37°C. 2A- represents the baseline. 2B- Final concentration of 10 μM ATP application, after a period of 2 minutes in saline. The arrows represents the moment of agonist application.2C- 10 μM ATPγS application. 2D- 10 μM UDP and 2E- 10 μM β,γ-meATP. All recordings were performed at distinct cells. (2F) Bar graph representation of 3 independent experiments. *** p<0.01 when baseline (saline) was compared to UTP treatment. ** or * p<0.05 when there was statistical significance between saline and some agonist listed. +++ p<0.01, ++ or + p<0.05 when there was statistical significance between UTP and some agonist listed.</p

Currents elicited by the activation of P2X receptors in eosinophils.

<p>All records were obtained at 37°C. All agonists were applied at concentration of 10 μM and we maintained the Pipette holding of (Vhold = -60 mV). (1A-L) Patch clamping whole cell recordings after application of P2 receptor agonists. Arrows indicate the point of agonist application. (1M) Bar graph representing the quantitative relation of the ionic currents amplitude normalized by cell capacitance, after agonist activation of P2X receptors (n = 5 independent experiments). *** p< 0.01 when ATP was compared to other agonists; +++ p<0.01 when ATPγS was compared to other agonists; $$ $ p<0.01 when 2MeSATP was compared to other agonists; ### p<0.01 when ADP was compared to other agonists; &&&p<0.01 when α,β-meATP was compared to other agonists; @@@ p<0.01 when β,γ-meATP was compared to other agonists; § p<0.05 when BzATP was compared to other agonists.</p

Immunofluorescence assays for P2 receptors in eosinophils.

<p>Cells were fixed in a quantity of 10⁵ cells per experiment. The immunofluorescence was done against P2XR and P2YR, with rabbit primary antibodies (Alomone Labs), and then a secondary antibody conjugated to Alexa red 546 was added to the preparation and visualized in the fluorescence microscope. We had positive reaction for P2X1, P2X₂, P2X₄, P2X₇ and P2Y₁, P2Y₂, P2Y₄.These images were obtained with objective of 40X.</p