To generate trypanosome fractions, a suspension titrating 4×108 parasites per ml, obtained by DEAE-cellulose chromatographic isolation, was repeatedly freeze-thawed, thoroughly vortexed until no intact trypanosome could be detected under the microscope, and centrifuged for 30 min at 14 000 g and 4°C to separate the cytosolic and non-cytosolic fractions.
In brief, material from HeLa cells infected under different conditions and times was harvested from wells using a cell scraper, frozen at −80°C, thawed and thoroughly vortexed before titrating on 50% confluent HeLa cells for quantification by light microscopy or immunofluorescence on a widefield fluorescence microscope (Leica, Deerfield, IL, USA).
For DNA extraction, each sample was thawed and thoroughly vortexed, swab was removed and the solution was transferred to a 1.5 ml tube containing Phase Lock Gel Light (Eppendorf, Hamburg, Germany).
At the time of assay, the samples were thawed, mixed thoroughly, and diluted 1 25 with 0.1 M NaPO4 buffer (pH 8.0) without Triton X-100 for a final 1 250 dilution of sample.
Briefly, the tubes containing the swabs were thawed and thoroughly homogenized by vortexing, and 140 μl of the medium were added to 540 μl of the RLT buffer that is included in the kit, followed by 700 μl of 70% ethanol, and then transferred to a Mini RNeasy column previously inserted into a 2-ml collecting tube.
This mixture was subjected to three cycles of freeze thawing, vortexed thoroughly and centrifuged at 20000 g for 30 min at 10°C.
Once the ground is thoroughly thawed, there they are, tugging on earthworms as though they were the hawsers of the S.S. Earth.
It will be the fifth meeting between Francis and el-Tayeb, evidence that Al-Azhar's freeze in relations with the Holy See sparked by Pope Benedict XVI's 2006 comments linking Islam to violence has thoroughly thawed.
Daughter sets were thawed and mixed thoroughly, before 1 μl was transferred to an empty 96-well tissue-culture plate (Greiner) using a Platemate 2X2 liquid handler (Matrix).
When used, aliquots were slowly thawed in ice, thoroughly mixed and centrifuged for 5 min at 16000 × g to remove precipitate if present.
