For this assay, healthy P. stylirostris shrimp were injected with the infected medium in 10% volume of their corporal mass in the third abdominal segment.
10 bacteria were spotted onto this swimming medium in a volume of 5 μL and the intensity of the halo of swimming was measured after 24 h of incubation at 37°C.
Muscle fibers were then extracted by trituration after dilution of the digestion medium in one volume of DMEM.
Solvent solutions or Apis mellifica preparations were added to the culture medium in a volume of 5 μL in a total incubation medium of 0.5 mL, obtaining a 0.3% final ETOH concentration, which did not have any cytotoxic effect.
In addition, in some experiments, MCF-7 cells and pII, PC-3, MDA-231 and HBL-100 cells were seeded in flat-bottomed 96-well tissue culture plates in triplicate at a concentration of 1 × 10 cells/ml medium in a volume of 100 μl per well and allowed to grow to 70% confluency before the addition of asiaticoside.
To examine the effect of MRS components on the ability of the recombinant Scaf · AT scaffoldin to bind to the cellulose-coated slide, 0.5 mg/ml of the scaffoldin was subjected to 1-hour interaction at room temperature with each component concentrated as in Lactobacillus-MRS bacterial growth medium in a volume of 1 ml.
For tissues (n = 14) weighing more than 0.5 gram (TB71 - TB148) the tissue was minced as described above and then enzymatically dissociated in tissue digestion buffer containing collagenase I (Worthington), hyaluronidase (Sigma), Collagenase IV (Worthington) at 1 mg/ml of each enzyme in DMEM/F12 medium in a volume of 1 5 ratio of tumor to buffer (wt/vol) on a gyrating platform at 37o C for 30 min.
APE was added to the culture medium in a maximal volume (<0.4%) and the same volume of ethanol as the solvent was added to the medium of the control cells.
CD14+ cells were seeded 1·105 cells per well in a 96-well plate and stimulated for 24 hr with 100 ng/ml LPS, in combination with 100 µl supernatant of CD56+ cells, 2.5 ng/ml IFN-γ (Biosource) or medium in a final volume of 200 µl.
All cells were plated into complete medium in a total volume of 200 μl.
