Lmp agarose

A. vaga individuals were dried following the optimized protocol previously described in [16 (link)]. Briefly, cultures were washed with 15 mL Spa® water the day before their collection. Individuals were detached from the petri dish with a cell scraper followed by a short round of vortex. Animals were transferred to a 15-mL Falcon tube for centrifugation. Pellets, from each petri dish, were pooled in a final tube. The concentrated pellet containing rotifers was resuspended in Spa® water to a concentration of 80.000 individuals per mL. Then, for each desiccation sample, 0.5 mL of liquid was placed in the center of a Petri dish containing 30 mL of 3% Low Melting Point agarose (LMP agarose, Invitrogen). LMP agarose plates with hydrated individuals (approx. 40,000 per plate) were placed in a climatic chamber (WEKK 0028) for dehydration with the following parameters: (1) linear decrease in relative humidity (RH) from 70 to 55% for 17 h (Temperature: 21–23 °C), (2) linear decrease in relative humidity from 55 to 41% for 1 h (Temperature: 21–23 °C), and (3) maintenance at 41% RH and 21 °C for the desiccated period. Based on this protocol, it took approximately 37 h after the start of the dehydration process to dry all A. vaga individuals.

In order to check the clonogenic ability of U87MG, cells were harvested 48 h post-siRelA/siControl transfection. A total of 15,000 cells/well were suspended in 0.3% LMP agarose (Invitrogen) in DMEM supplemented with 20% FBS and seeded on top of 0.6% agarose in 6-well plates in triplicates. 500 μl of complete medium was added on top of the agarose layer twice a week. After 22 days in culture, the colonies were stained using 0.2% crystal violet. Mean number of colonies with ≥30 μm diameter was counted microscopically at 4x magnification in ten non-overlapping fields per well.

VSV-KFDV vaccine vectors were serially diluted from 10⁻² to 10⁻⁹ and used to infect the confluent VeroE6 cells seeded in 6-well plates in duplicates (0.5 ml/well). Plates were incubated for 1 h at 37 °C while rocking. Subsequently, the inoculum was removed, and the cells were overlayed with 2 ml of a 1:1 mixture of 2% LMP agarose (Invitrogen, Carlsbad, CA) in 2 × MEM/2% FBS (Thermo Fisher Scientifc). The agarose was allowed to solidify, and cells were incubated for ~72 h at 37 °C. The plaques were stained by crystal violet solution and the titer for each vaccine was calculated in PFU/ml.

Plaque assay was performed to determine titers of the virus stocks and supernatant samples as described elsewhere with minor modification (Zhou et al, 2017 (link), 2018 (link)). Briefly, MDCK cells were seeded in 12‐well plates. Confluent monolayers were inoculated with 400 µl of 10‐fold serial dilutions of samples and incubated for 1 h at 37°C. After removing the inoculum, the monolayers were overlaid with 1% LMP Agarose (Invitrogen) supplemented with MEM and 1 µg/µl TPCK‐treated trypsin and further incubated for 2–3 days. The monolayers were fixed with 4% PFA and stained with 1% crystal violet to visualize the plaque after removing the agarose plugs. Virus titer was calculated as plaque‐forming units (PFU) per milliliter.

Samples were mounted in 1% low melting point (LMP) agarose (Invitrogen) and imaged with a Leica SP5II confocal microscopy (Leica LAS software) using 10x PL APO CS (dry), 20x immersion lens (phalloidin) or 40x PL APO CS (oil) lenses (cristae imaging). LasX (Leica) and Amira 6.0 (FEI) was used for 2D and 3D rendering, image analysis and picture acquisition.