Supplementary MaterialsSupplementary material mmc1. hydrogen peroxide. Fresh pictures appear just as these were captured over the microscope, while prepared pictures present the binarization supplied by software employed for measurements of mitochondrial morphology. For in-depth debate from the tests and computational strategies regarding this data, please make reference to the corresponding analysis article titled Completely automated software program for quantitative measurements of mitochondrial morphology (McClatchey et al., in press) . Specs Table Subject region em Biology /em Even more specific subject region em Mitochondrial dynamics /em Kind of data em Pictures /em How data was obtained em Microscopy and picture handling /em Data structure em PNG /em Experimental elements em HUVEC cells, principal SMCs from GK and Wistar rats, and SH-SY5Y neurons cultured for 6C8 passages in MCC950 sodium supplier vitro. /em Experimental features em Set cells stained for Tom20 (mitochondria), nitrotyrosine (cytoplasm), and DAPI (nuclei) and binarized using computational strategies /em , , Data supply area em Aurora, Colorado, United states /em Data ease of access em Data is at this post. /em Open up in another window Worth of the info ? Investigators considering usage of this mitochondrial morphology dimension technique can make reference to these pictures for evaluation of software program quality.? Visitors to whom the endpoints reported in the connected primary MCC950 sodium supplier study content  are interesting may use these pictures as a Rabbit Polyclonal to PBOV1 visible reference.? Investigators taking into consideration usage of this mitochondrial morphology dimension technique can evaluate their pictures to these to assess whether this software program does apply. 1.?Data The pictures shown here contain raw microscope pictures (still left) and software-binarized pictures (ideal) acquired or generated for person cell culture tests in the associated major study content . The 1st picture acquired for every treatment group can be shown; please make reference to the Supplementary components for the entire set of uncooked microscope pictures. The info included above (i.e. specs table, worth of the info, data description with this paragraph) pertains to all picture models below. Experimental style, components and strategies are contained in both general and experiment-specific conditions. 2.?Experimental design, materials and methods 2.1. Reagents Dulbecco?s Modified Eagles Medium (DMEM) 5?mM and 25?mM glucose and non-essential amino acids and Laughlins F12 Medium were obtained from Thermo Scientific Hyclone, and trypsin and trypsin inhibitor were purchased from Fisher Scientific. Fetal bovine MCC950 sodium supplier serum (FBS) was procured from Gemini Bioproducts. Hank?s Balanced Salt Solution (HBSS) was purchased from Corning Life Sciences. Secondary detection antibodies Alex Fluor 488 and 546?were purchased from Life Technologies. Antibodies to TOM20 (rabbit) and nitrotyrosine (mouse) were procured from Santa Cruz Biotechnology. 2.2. Cell culture Primary rat vascular smooth muscle cells (SMCs) were cultured in low glucose (5?mM) DMEM with 10% fetal bovine serum (FBS), 1% L-glutamine, 1% non-essential amino acid blend, and 1% Pen/Strep, all expressed as % by volume. For SMC starvation media, the same proportions were maintained, except that FBS was reduced to 0.1%. Human umbilical vein endothelial cells (HUVECs) were cultured in F-12k medium (Hyclone #SH30526.01) with 10% FBS, 1% Pen/Strep, 0.05?mg/mL endothelial cell growth supplement, and 0.1?mg/mL heparin. SH-SY5Ys (ATTC neuronal cell line) were cultured in 45% F-12k medium, 45% MCC950 sodium supplier low glucose DMEM, and 10% FBS. All cells were cultured at 37?C at 5% CO2. Media was changed at least every three days, and cells were split 2:1 at 80% confluence. Experiments were performed on cells at 50C70% confluence. Unless otherwise specified, cell culture materials were obtained from Santa Cruz Biotechnology. 2.3. Fixation and staining Prior to fixation, phosphate buffer solution (PBS) rinse and paraformaldehyde solutions were warmed to 37?C (except as otherwise indicated). Samples were washed 3 in warm PBS prior to incubation in 4% paraformaldehyde at 37?C for 15?min. Pursuing fixation, samples had been cleaned 3 in warm PBS and quenched in 50?mM NH4Cl for 15?min in room temperature. Samples then were.