Bio gen pro200 homogenizer

DNA extraction assay was performed using DNEasy Blood and Tissue kit (QIAGEN, Germany), following the manufacturer’s procedure provided in the kit. Briefly on tissue preparation, approximately 25 μg of tissue from the back of the triatomine was cut with a leaf of new scalpel for each sample. The cut portion of the tissue was placed in a 1.5 ml tube with 200 μl of tissue lysis buffer (Buffer ATL, accompanied in the mentioned kit), followed by maceration with a homogenizer (Bio-Gen PRO200 Homogenizer, Pro Scientific, USA).
The PCR was conducted using 121 and 122 primers (121: 5′-AAA TAA TGT ACG GG(T/G) GAG ATG CAT GA-3′, 122: 5′-GGT TCG ATT GGG GTT GGT GTA ATA TA-3′) targeting specifically the kinetoplast minicircle DNA sequences [10 (link), 11 (link)]. Each reaction was performed in a total of 25 μl reaction volume containing 0.5 μmol/L of each primer, 12.5 μl of GoTaq DNA polymerase (Promega, USA), 5 μl of DNA template, and additional distilled water to the final reaction volume. Briefly, the PCR condition was: 3 min at 94 °C for the denaturation, followed by 35 cycles of 30 s at 94 °C, 30 s at 55 °C, and 30 s at 72 °C with a final extension of 10 min at 72 °C. The amplified PCR product was detected at the size of 330 bp in the electrophoresis using 3% of agarose gel.

To induce the LH surge^{43 (link),67 (link),68 (link)}, female C57BL/6 mice underwent bilateral ovariectomy. LH surge was induced through one of two paradigms. For most animals, five days after surgery, animals were given a subcutaneous injection of 0.25 ug of β-estradiol benzoate (EB; Sigma-Aldrich) in 100 μl of sesame oil 4 h after lights on. On the following day, animals were given 1.5 μg of EB in sesame oil (100 μl) 4 h after lights on. The following day, animals were euthanized at lights off and pituitaries were snap frozen on dry ice and blood collected and processed to serum. Sera was processed using a Luminex assay and the Millipore MAGPIX as described, and an LH surge was considered a value greater than 1.5 ng/ml. The LH surge in three animals was induced by ovariectomy and E2 pellets as described^{43 (link)}. There is no difference in the amplitude of the LH surge between these paradigms^{43 (link)}. For this paradigm, LH in serum samples was measured via Luminex or via the University of Virginia Ligand Core, and an LH surge was accepted at a concentration of 0.6 ng/ml. Pituitaries where homogenized (PRO Scientific Bio-Gen PRO200 Homogenizer), then lysed in RIPA/NP-40 lysis buffer. Western blots were performed on pituitary lysates.

A murine pulmonary infection model was used to evaluate the virulence of P. aeruginosa strains. Briefly, bacterial cells of overnight culture were washed three times and re-suspended in phosphate-buffered saline. Bacterial suspensions were administrated intranasally to each mouse at 20 µl per mouse under anaesthesia (approx. 1 × 10⁶ CFU per mouse). After 24 h, all mice were sacrificed and the lungs were harvested and kept in ice-cold 0.9% NaCl saline. The bacterial cells residing in the lungs were suspended into the 0.9% NaCl saline by homogenization using a Bio-Gen PRO200 Homogenizer (Pro Scientific). CFU was quantified by serial dilution and plating on LB agar.