Omni bead ruptor 24 bead mill homogenizer

SW1088 cells (transduced with EV or ATAD1-FLAG) were grown under normal culture conditions, as described above. Cells (3×10⁶) were mixed 1:1 with Matrigel (Corning) and injected into one flank per mouse. Mice were male NOD/SCID aged 13–15 weeks. Tumor volumes were monitored biweekly using a Biopticon TumorImager. Animal experiments were conducted in accordance with The University of Utah IACUC.
PC3 cells (transduced with LentiCRISPRv2-sgNT or sgATAD1) were grown as described above. Cells (1.8×10⁶) were mixed 1:1 with Matrigel (Corning) and injected into one flank per mouse (12-week-old, male, NRG). Once tumors had established, mice were randomized into bortezomib or vehicle groups and treatment occurred by tail-vein injection twice weekly for 4 weeks. At the conclusion of the experiment, mice were sacrificed within 24 hr of receiving an IV injection. Tumors were harvested and snap-frozen on liquid nitrogen. Tumor fragments were homogenized in RIPA buffer using ceramic beads on a Omni Bead Ruptor 24 Bead Mill Homogenizer (2 cycles of 45 s at 6 m/s, 4°C). Homogenates were centrifuged (16,000 RCF, 10 min, 4°C) and supernatants were recovered, then used for downstream analysis by immunoblot. Data were analyzed by mixed effects model with Tukey’s multiple comparisons. Animal experiments were conducted in accordance with The University of Utah IACUC.

Different assays were employed to diagnose and genotype the causative agent behind this outbreak. For diagnosis, a one-tube, semi-nested PCR (snPCR) was performed to detect CyHV-3 in certain fish tissues, namely the skin, gills, liver, kidney, heart, spleen, brain and intestines. The PCR products of this assay were then sequenced.
For genotyping, four PCR protocols coupled with sequencing were performed.
Genomic DNA from fish tissues were isolated as follows: fish tissues were cut into small pieces and then homogenized using an Omni Bead Ruptor 24 Bead Mill Homogenizer (Omni International, USA). Genomic DNA was extracted from the above-mentioned tissues using DNeasy Blood & Tissue Kits (Qiagen, Germany) according to the manufacturer's instructions. A diagnostic one-tube snPCR was used to detect the CyHV-3 virus, and this PCR protocol, which targeted the CyHV-3 major glycoprotein gene (ORF 56), was performed according to a previously published study [12 (link)]. Positive results in the snPCR showed three bands with sizes of 464, 372, and 182 bp. Similarly, to detect the presence of carp edema virus, a nested PCR protocol was performed to target a partial sequence from the 4a gene according to a previously published study [13 (link)].