Lcachn 40x php

Acanthamoeba locomotion and intracellular motion were observed with an inverted phase contrast microscope (Olympus CKX41 and IX81, Olympus Deutschland GmbH, Hamburg, Germany) with either a LCACHN 40X PHP, a LUCPLANFLN 40X PH2 or a UPLFLN 60X (long distance) objective (all from Olympus). Sequences having a length of at least 30 sec were recorded with a high-speed camera (Hamamatsu C9300, Hamamatsu, Japan) at frame rates between 80 and 99 frames per second (fps). Exposure times chosen for image recording are not expected to cause errors in the analysis of particle trajectories60 (link).

For inhibiting myosin II motors, (-)-blebbistatin was used. The reagent binds to ATPase, which is a catalyzer for ATP hydrolysis and hence blocks the myosin heads in their unbound state as it slows down ATP hydrolysis, especially the release of phosphate45 (link). (-)-blebbistatin (Sigma-Aldrich, Germany) was dissolved in DMSO (Roth, Germany) at 1 mM concentration. (-)-blebbistatin was added to the medium of the cultured acanthamoebae to reach different concentrations (50 μM, 60 μM, and 70 μM). Right after the addition of (-)-blebbistatin, time-lapse imaging was started. Here, we employed for the experiments a phase contrast microscope (Olympus IX81, Olympus Deutschland GmbH, Hamburg, Germany) with a 40 × (LCACHN 40X PHP) objective. After a period of 15 min to 1 h the effect of (-)-blebbistatin decreased and time-lapse sequences were recorded again to prove the viability of the cells by monitoring intracellular motion and locomotion. Acanthamoebae had completely normal appearance after 1–2 h of relaxation from the drug treatment.