Nb 4g

The surgically removed X. laevis oocytes were defolliculated by treatment with collagenase (NB 4G, Serva, Heidelberg, Germany). Oocytes at Dumont stages V–VI were injected with 23 or 46 nl of cRNAs diluted to 0.1 to 1 µg/µL for hP2X5^FL or 0.05 µg/µL for hP2X7, respectively, to keep the maximal ATP-elicited current amplitudes < 5 μA to limit the current-induced stress for the oocytes. The oocytes were incubated at 19 °C in a sterilized frog Ringer’s solution (Mg/Ca-ORi: 100 mM NaCl, 2.5 mM KCl, 1 mM MgCl₂, 1 mM CaCl₂, and 10 mM HEPES, pH 7.4) and supplemented with penicillin (100 U/mL) and streptomycin (100 μg/mL).

All animal experiments were approved by the federal state authority of Saxony (reg.no. TVV15/16) and followed all legislation of the animal welfare act. Immune-deficient Pfp/Rag2^−/− (C57BL/6N(B6.129S6-Rag2(tm1Fwa)Prf1(tm1Clrk))) and corresponding wildtype C57BL/6N male and female mice were housed under standard conditions with a 12-hour circadian rhythm at ambient temperature with free access to food (chow diet, V1534, Ssniff, Soest, Germany) and water.
Animals were matched for age, body weight and sex (Table 1). The following abbreviations are subsequently used for the groups (a) male young mice: MY, (b) female young mice: FY, (c) male adult mice: MA, (d) female adult mice: FA, (e) wildtype C57BL/6N: WT and (f) knockout Pfp/Rag2^−/−: KO (Table 1).
For all experiments, livers and adipose tissue were immediately frozen and stored at −80 °C, cryopreserved for cryosections, or fixed with 4% Histofix. Body, liver, and adipose tissue weight were measured and organ-to-body weight ratios were calculated. Blood samples were taken and serum was stored at −40 °C.
Primary hepatocytes were isolated from young and adult, female and male wildtype and knockout mice (cf. Table 1) after anesthesia with Narcoren^®, performing a two-step liver perfusion with collagenase (NB4G, Serva Electrophoresis GmbH, Heidelberg, Germany) essentially as described [23 (link),24 (link)].