Minimal essential medium eagle

The study made use of previously described cultured Boa constrictor kidney cells, I/1Ki, [34 (link)] and persistently SwSCV-1- infected I/1Ki cells, I/1Ki-Δ [17 (link)]. The cells were maintained in Minimal Essential Medium Eagle (Sigma-Aldrich, St.Louis, MO, USA) supplemented with 10% fetal bovine serum (ThermoFisher Scientific, Waltham, MA, USA), 200 mM L-glutamine (Sigma-Aldrich, St. Louis, MO, USA), 100 µg/mL of streptomycin (Sigma-Aldrich, St. Louis, MO, USA), and 100 U/mL of penicillin (Sigma-Aldrich, St. Louis, MO, USA) in an incubator at 30 °C with 5% CO₂.
To establish another persistently SwSCV-1-infected I/1Ki cell line, designated I/1Ki-1.2×Δ, we transfected I/1Ki cells with a plasmid containing 1.2 copies of the SwSCV-1 genome (described below) and maintained the cells as described above and earlier [17 (link)].
To study infectious particle formation of the SwSCV-1-infected cell lines, we conducted superinfection studies with HISV-1 [35 (link)], earlier demonstrated to be an efficient helper virus for SwSCV-1 [17 (link)]. The superinfection studies and detection followed the protocol described [17 (link)]. For the infection, we used 600 copies of HISV-1 S segment RNA per cell, which corresponds roughly to a multiplicity of infection (MOI) of 10.

Human neuroblastoma cell line, SH-SY5Y(ATCC CRL-2266), was grown and maintained in a 1:1 mix of Minimal Essential Medium Eagle (Sigma) and Nutrient Mixture F-12 Ham (Sigma), supplemented with 10% foetal bovine serum (ThermoScientific), 100 U/ml penicillin/100 mg/ml streptomycin (Sigma), 1% (v/v) 200 mM l-glutamine (Sigma), and 1% (v/v) 100 mM sodium pyruvate (Sigma). Cells were incubated at 37 °C with 5% CO₂.
Cells were plated out into 24-well plates for reporter gene transfections or 6-well plates to assay endogenous gene expression. For drug challenge, 10 μM cocaine, 1 mM lithium, or vehicle alone (sterile water) were diluted in appropriate volumes of cell culture media and added to the SH-SY5Y cells for 1 h, removed and fresh media added. For luciferase assays, drug treatments were performed 4-hour post-tranfection.

The established neuroblastoma cell line SH-SY5Y (ATCC: CRL-2266) was derived from cell line SK-N-SH, which was originally extracted from bone marrow metastasis of 4-year-old female with neuroblastoma. SH-SY5Y cells were incubated at 37 °C and 5% CO₂ in a 1-to-1 mix of Minimal Essential Medium Eagle (M2279, Sigma-Aldrich/Merck, Darmstadt, Germany) and Nutrient Mixture F-12 Ham (N4888, Sigma-Aldrich), supplemented with 10% (v/v) FBS (10500-064, Gibco/ThermoFisher Scientific, Waltham, Massachusetts, U.S.), 1% (v/v) penicillin–streptomycin (P0781, Sigma-Aldrich), 1% (v/v) L-glutamine (25030-149, Gibco), and 1% (v/v) 100nM sodium pyruvate (S8636, Sigma-Aldrich).

Donated human spermatozoa with normal semen characteristics (concentration, motility; WHO 2010) from healthy volunteers with no known fertility problems were used in this study. Samples were obtained by masturbation after ≥ 48 h of sexual abstinence, liquefied for 30 min at 37°C, and processed using density gradient centrifugation, to separate cells using 40/80% Percoll (Sigma Aldrich, UK) fractions. Every experiment has been performed on pooled spermatozoa from ≥ 3 donors each.
For experiments in non-capacitating conditions, we used a buffer system slightly modified from [23 (link)] using Minimal Essential Medium Eagle (Sigma), supplemented with HEPES (1 M solution, Gibco), sodium lactate (DL-solution, Sigma), sodium pyruvate (100 mM solution, Gibco) and bovine serum albumin (7.5% solution, Sigma).
For experiments in capacitating conditions, we used a buffer system slightly modified from the HTF from [63 (link)] to a final composition of 97.8 mM NaCl, 4.69 mM KCl, 0.2 mM MgSO4, 0.37 mM KH2PO4, 2.04 mM CaCl2, 0.33 mM Na-pyruvate, 12.4 mM Na-lactate, 2.78 mM glucose, 21 mM HEPES, 25 mM NaHCO₃ and 3 mg/mL BSA. All buffer components were supplied by Sigma-Aldrich unless otherwise stated and all buffer systems were adjusted to pH 7.4.

Beas2B and A549 cells (ATCC) were grown in Dulbecco's Modified Eagle's Medium (DMEM, Sigma-Aldrich), HeLa cells (ATCC) were grown in Minimal Essential Medium Eagle (Sigma-Aldrich), and H1299 and H1975 cells (ATCC) were grown in Roswell Park Memorial Institute-1640 Medium (Sigma-Aldrich). All media were supplemented with 10% FBS, 4 mM L-glutamine, and 1% Penicillin/Streptomycin, and cells were incubated at 37°C in a 5% CO₂ incubator. Please note that our strain of Beas2B cells does not cause tumors in nude mice [60 (link)].

Minimal essential medium eagle (MEME) was purchased from Sigma-Aldrich (Munich, Germany). Other cell culture reagents were obtained from Biochrom (Berlin, Germany) or life Technologies (Darmstadt, Germany).