Lithium heparin microvettes

Males at 11 w of age were used for testosterone analysis. The animals were either group-housed at six animals per cage or single-housed. After 24 h and 48 h of separation at 10 am, we collected 25 μL of blood from the tail in lithium/heparin microvettes (Sarstedt, 16.443) and the blood was spun down at 2500 g for 10 min at 4 °C to obtain plasma. We analyzed the testosterone levels using testosterone ELISA measurements according to the manufacturer’s protocol (Biovendor, RTC001R).

For fluorescent Zn-staining of cultured cells, growth medium was discarded and the cells were washed with PBS. Coverslips were incubated with a solution of 50 μM Zinpyr-1 (Burdette et al. 2001 (link)) in PBS for 1 h at RT. The mean intracellular Zn concentration per cell was determined by Zinpyr-1 fluorescence intensity. Images were obtained using an Axioscope microscope and evaluated with ImageJ 1.50 g. The Zn-concentration of solutions and blood was measured by flame atomic absorption spectrometry (AAS) at the Department of Clinical Chemistry (ZE klinische Chemie) of the University Hospital Ulm using a PinAAcle 900T from Perkin Elmer and in the workgroup of Prof. Rink, Institute of Immunology, University Hospital Aachen using a Perkin Elmer AAnalyst 800 instrument. All reagents for atomic absorption were of appropriate quality for trace element analysis (TraceSelect, Fluka). Samples were diluted in ultrapure water containing 0.2% (v/v) HNO₃ and quantified with a standard curve between 0 and 1 mg l⁻¹ zinc in 0.2% (v/v) HNO₃, measuring the absorption at 213.9 nm (slit 0.7 nm). A reference samples spiked with defined concentrations of Zn were routinely analyzed to ensure reliable quantification. Whole blood from mice was collected in Lithium-Heparin Microvettes (Sarstedt) and stored at −20 °C until further analysis.