Low melting agarose

For transmission electron microscopy (TEM) analysis, 1 × 10⁸ sperm cells suspended in Tyrode’s albumin lactate pyruvate media [22 (link)] were inoculated with ASFV “ArmeniaΔ285L-GFPhuCD4” [14 (link)], gt II (MOI 5). Sperm cells were incubated with the virus suspension (48 h, 37°C, 5% CO₂, humidified atmosphere). A negative sperm cell culture control was treated similarly to verify appropriate handling and culture conditions.
Subsequently, inoculated sperm cells were washed three times in 1× PBS by centrifugation at 134× g, 10 min, 23 °C. The resulting pellet was treated with fixing solution (2.5% glutaraldehyde buffered in 0.1 M sodium cacodylate (pH 7.2), 300 mosmol, Serva Electrophoresis, Heidelberg, Germany) for at least 2 h at 4 °C, and embedded in 1.8% low-melting agarose (Biozym). Small pieces were postfixed in 1.0% aqueous OsO4, and stained en bloc with uranyl acetate. After stepwise dehydration in ethanol, cells were cleared in propylene oxide, infiltrated with Glycid Ether 100 (Serva Electrophoresis), and polymerized at 60 °C for 3 days. 60–70 nm ultrathin sections were prepared with an ultramicrotome (UC7, Leica Microsystems, Wetzlar, Germany) and collected on EM grids (300 mesh, Plano). Finally, the sections were counterstained with uranyl acetate and lead citrate and analyzed with a Tecnai-Spirit TEM (FEI) at an accelerating voltage of 80 kV.

Anchorage-independent growth was assessed as previously described [21 (link),22 (link)]. Briefly, 6-well plates were coated with a base layer of 0.9% low-melting agarose (Biozym Scientific GmbH, Hess. Oldendorf, Germany) containing 10% FBS, antibiotics and the respective reagents or corresponding solvents. A layer of 0.35% agarose containing the same supplements as the base layer plus 2 × 10⁴ cells/well was placed on top of the base layer prior to incubation for 21 d at standard culture conditions. Microscopic images were taken at 4-fold magnification. The largest diameter of the colonies was measured, and colonies with a diameter exceeding 150 µm (T98G, SC35, SC38 and SC40) or 200 µm (U87MG, PC35, PC38 and PC40) were counted.

Cells were seeded (4*10⁵/well) in 6-well plates, grown overnight and exposed to 500 ng/ml NCS for 1 h. As positive control, cells were treated with 50 µM tert-Butyl hydroperoxide (B2633, Sigma-Aldrich). Cells were harvested, mixed 1:10 with 0.5% low melting agarose (Biozym) and plated on microscopic slides, pre-coated with 1% regular agarose (Biozym). Slides were incubated in cold lysis buffer (2.5 M NaCl, 100 mM EDTA, 1% Triton X-100 and 10 mM Tris pH 10) at 4 °C for 1 h. Lysis buffer was removed and exchanged to cold alkaline electrophoresis buffer (300 mM NaOH, 1 mM EDTA pH 13). After a 25 min incubation at 4 °C, the electrophoresis was conducted at 1 V/cm (22 V) at 4 °C for 15 min. The slides were washed with water, fixed with 70% EtOH and air-dried. The DNA was stained with Vista Green DNA staining solution (238,554, abcam) and DNA comets were inspected by microscopy. The DNA comets were analyzed using the OpenComet plugin [58 (link)] for imageJ. The olive moment was used to compare the conditions. Significant differences between groups were identified with the Kruskal–Wallis rank sum test in R. Pairwise comparisons were investigated performing the Wilcoxon rank sum test and p values were corrected using the Bonferroni method.