Pcr tubes

Single cells of G. semen were isolated manually under a stereomicroscope (50–80× magnification) using micropipettes custom-made from microcapillaries (minicaps 100 µl; Hirschmann). Isolated cells were washed twice in 2 µl droplets of sterile filtered (0.2 µm) MWC + Se culture medium (Gollnisch et al. 2021 ) and once in 2 µl droplets of 1× phosphate-buffered saline (PBS) buffer pH 7.4 (Gibco) on sterile 96-well plate lids. Both medium and buffer were decontaminated through UV treatment at 254 nm wavelength using a UV crosslinker (Spectrolinker) before use. Individually washed single cells were transferred into PCR tubes (Sarstedt) in 1 μl PBS, using a new glass capillary for each isolation and each transfer to avoid cross-contamination. All single-cell isolates were frozen immediately and were stored at −80 °C until amplification.

All chemicals (p.a. grade), cell culture medium, and supplements were obtained either from Carl Roth GmbH (Karlsruhe, Germany) or Sigma-Aldrich Chemie GmbH (Taufkirchen, Germany) with the exception of fetal bovine serum (FBS), which was bought at Thermo Fisher Scientific GmbH (Dreieich, Germany). Cell culture dishes and flasks, reaction tubes, PCR tubes, and other consumables were purchased from Sarstedt (Nuembrecht, Germany). CuO NP were kindly provided by Dr. Wendel Wohlleben (BASF SE, Germany) in the course of the BMBF-funded project MetalSafety and originally synthesized and obtained from by PlasmaChem (Berlin, Germany). This particle species was also previously examined in the EU FP7-Project SUN, including oral and inhalation in vivo studies [28 (link),60 (link)]. Primer pairs were synthesized by Eurofins (Ebersberg, Germany). DNA suspension buffer, PCR certified water, and TE buffer were obtained from Teknova (Hollister, USA). The 2× Assay Loading Reagent, 20× DNA Binding Dye, and IFCs were purchased from Fluidigm (San Francisco, USA). The 2× SsoFast EvaGreen Supermix was provided by Bio-Rad (Munich, Germany), and the 2× TaqMan PreAmp Master Mix was bought from Applied Biosystems (Darmstadt, Germany). Exonuclease I was obtained from New England Biolabs (Frankfurt am Main, Germany).