Mean number and length (m) of neurites were calculated by Neurolucida software (Version 8, MBF Bioscience, USA), respectively
Mean number and length (m) of neurites were calculated by Neurolucida software (Version 8, MBF Bioscience, USA), respectively. magnetic stirring also exhibited comparable neuron-specific protein expression to those in the absence of oA42. The phagocytic uptake of mpA42 by microglia was enhanced significantly as compared to the counterpart of oA42, and the M1 polarization of microglia often occurring after the uptake of oA42 restricted to an appreciable extent. As a result, the inflammation induced by pro-inflammatory cytokines was greatly alleviated. = 10). The morphology of mpA42 was examined by TEM, scanning Ivachtin probe microscopy (SPM, tapping mode, Bruker, Model-Dimension Icon, Camarillo, CA, USA) and atomic pressure microscope (AFM, Bruker, Santa Barbara, CA, USA) [29,30,31,32]. 2.5. Cell Membrane Damage and Neurite Outgrowth Impairment 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and trypan blue assays were used to determine the cell viability of N2a cells co-incubated with oA42 species (160 M) under the MSB stirring treatment (2500 rpm, 2 h). For 160 M oA42 preparation, the peptide film obtained previously was dissolved in dry DMSO to a concentration of 5 mM. The solution was then diluted with the DMEM medium (phenol red-free) to an A42 concentration of 160 M, followed by re-incubation at 4 C for 24 h. N2a cells were first seeded into 96-well plates at a concentration of 5 103 cells/well in 200 L DMEM, made up of 10% fetal bovine serum (FBS) and 1% penicillin, and incubated at 37 C overnight in an atmosphere of 5% CO2. N2a cells were then co-incubated with oA42 (160 M) with or without MSB-based (144 g mL?1) stirring at 2500 rpm for another 2 h. After being washed twice with PBS, Rabbit polyclonal to CD105 Ivachtin cells were re-incubated in DMEM Ivachtin for another 24 h. MTT (0.25 g/mL) in DMEM (200 L) was added into each well, followed by re-incubation at 37 C for 4 h. The culture medium was then replaced with DMSO (100 L) and the absorbance of each well at 570 nm was determined by the microplate reader. The MTT assay for the cell viability evaluation was performed based upon the measurement of the cellular metabolic activity, in particular with respect to the activity of mitochondria reductase (dehydrogenase) by colorimetric determination. Trypan blue assay was used to determine the quantity of viable N2a cells. In brief, trypan blue answer (0.4%) in DMEM (200 L) was co-incubated with N2a cells being treated with either oA42 (160 M) alone or oA42/magnetic stirring (2500 rpm, 2 h) for 24 h at 37 C. The viable N2a cells with intact cell membranes that prevent trypan blue from permeation into cells was then examined by optical microscopy. The effects of oA42 and mpA42 on neuron apoptosis in terms of the level of cell membrane damage was evaluated by measuring the release of LDH, a cytosolic enzyme from your damage cells . The neurite outgrowth was examined by laser scanning confocal microscope (LSCM, ZEISS LSM-780, Jena, Germany). The cell nuclei and cytoskeletons were stained with Hoechst 33342 and CytoPainter F-actin Staining Kit-Green Fluorescence, respectively. Mean number and length (m) of neurites were calculated by Neurolucida software (Version 8, MBF Bioscience, USA), respectively. The mean neurite length was defined herein as the ratio of the sum of neurite length to the number of neurite. At least three impartial experiments were performed in this study, and at least 50 cells quantified per experiment. 2.6. Cell Functions The cellular functions in terms of specific protein expression were examined by western blotting. Proteins were subjected to a 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene difluoride (PVDF) membrane. The PVDF membrane was blocked using 5% nonfat milk blocking buffer with anti-MAP-2, NF-M, and NeuN main antibody at 4 C for 24 h. It was then incubated at 37 C for 1 h with horseradish peroxidase-conjugated secondary antibody. -Actin was used as the loading control. The area and signal intensity of the detected bands were decided using the Molecular Imager PharosFX Plus System and quantified using the ImageJ software (v1.33, National Institute of Mental Health, MD, USA). For the LSCM examination, N2a cells on glass coverslips were treated with MSBs (144 g), free oA42 (160 M), and oA42/MSBs, respectively, either with or without 2 h magnetic stirring (2500 rpm) at 37 C. With additional 22 h incubation, the cells were washed with PBS.