A fine particle of A. blazei Murill (ABP), prepared by mechanical disruption, was used throughout the experiments.
Pelleted SDS-DTT treated worms were then digested with 100 µl of 15 mg/ml pronase (Sigma-Aldrich, St . Louis MO) in egg buffer at room temperature for 7 to 9 min. Mechanical disruption was applied during pronase digestion by pipetting the reaction up and down 60 times with a 1 200 µl tip pushing against the bottom of the 1.6 ml microfuge tube.
Unlike insecticides, the mechanical disruption is targeted specifically at the pest insects.
While this mechanical disruption is regarded for its high efficiency, high energy consumption and costs reduce the applicability of bead beating for microalgae cell disruption.
For example, the repair of epithelial monolayers following mechanical disruption is dependent on an intercellular calcium wave propagated from the site of disruption to adjacent cells [15], [16], [17], [18], [19].
Amongst the various cross-linking enzymes which protect proteins from proteolysis and mechanical disruption is transglutaminase 2 (TG2) (reviewed in [3,4]).
We then developed a workflow for single muscle fiber proteome analysis on the basis of a recent method, in which all preparation steps subsequent to mechanical disruption are performed in a single vessel, thereby minimizing sample loss (Fig 1A and Supplementary Methods section) [ 19].
The repair of the mechanical tissue disruption is the theoretical basis for the neo-vascularization process and subsequent pain relief following ESWT [ 8].
The L4-L6 spinal cords were rapidly dissected, treated with lysis buffer (DMEM/Accutase (PAA) 1 1, collagenase (3 mg/ml, Sigma), DNAse I (1 U/ml, Promega)) for 45 min at 37°C and subsequent mechanical disruption, which was done by forcing the tissue through a nylon mesh with 70 μm pore size (Cell Strainer, BD).
For mechanical disruption, cells were resuspended in 200 µl of SDS sample buffer. 1 volume of glass beads was added, and the samples beaten in a Precellys 24 bead beater (Stretton Scientific Ltd., 3 cycles of 30 seconds beating, 30 seconds pause).
Briefly, cell disruption was performed by a mechanical method adding an EDTA based lysis buffer.
