Alpha rvc

Plant materials were homogenized in liquid nitrogen and placed in an extraction mixture consisting of methanol/water/formic acid. The supernatants were evaporated in a vacuum concentrator (Alpha RVC, Christ, Osterode, Germany), and were then applied to a mixed mode reversed phase-cation exchange SPE column (Oasis-MCX, Waters, Milford, MA, USA), as previously described [52 (link)]. The cytokinin fraction was sequentially eluted, evaporated, and finally dissolved in 5% MeOH. An ultra-performance liquid chromatography (1290, Agilent, Pal Alto, CA, USA) coupled to a hybrid triple quadrupole/linear ion trapmass spectrometer (4500 Q TRAP, AB SCIEX, Waltham, MA, USA) was used to analyze each aliquot.

Insects alighting on or flying in the vicinity of the trunk, neck and head of the pasture group cattle restrained in headlocks were caught using a Japan insect hand net on td 24 at 1 and 2 pm, and on td 32 at 11 am. Insects were stored in 96.5% ethanol. Using a dissecting microscope, insect classification was conducted based on morphological characteristics at the Institute for Parasitology and Tropical Veterinary Medicine, Berlin, Germany. Using a vacuum evaporator (Alpha-RVC, Martin Christ Gefriertrocknungsanlagen GmbH, Osterode am Harz, Germany), insects were dried for 60 min at 37°C. DNA extraction was performed using the ZR Insect/Tissue DNA Kit-25™ (ZYMO Research Corporation, Irvine, CA, USA) following the manufacturer’s procedure with a variation in bead beater process time (15 min instead of 10 min). Every 11th sample, a negative processing control (empty tube), was included during DNA extraction. All DNAs were tested by real-time PCR using the Bb-RT2 assay as previously described [36 (link)].