Evos fl auto microscope

Between 50 and 80 μLs ultracentrifuged (at 100,000 × g) HUT102 cell supernatants (from 5-day old culture) at approximately 1 × 10⁹ EVs/mL were mixed with 1.5 μL of BODIPY^TM 493/503 (Cat. # D3922; Invitrogen^TM) to label EVs for 30 min at 37°C, and run on a Pharmacia G-50 spin column (1 mL bed volume in PBS buffer; 2000 rpm/2 min; Sorval RT6000D) to remove unbound dye. Resulting BODIPY EVs were quantified again by ZetaView and added onto recipient cells normalized to volume in biological triplicate. Treated cells were analyzed with an EVOS-FL-Auto microscope (Life Technologies), under 20× and 40× magnification with phase objective and fluorescence.

Golgi-cox staining was performed using the FD Rapid Golgi Stain Kit (FD Neurotechnologies). After dissection, brains were immersed in the mixture of solution A and B in the dark at room temperature for 14 days. Brains were then transferred to solution C and immersed for 7 days. Slices were sectioned at 120 μm with a vibrating microtome (Leica Microsystems, VT1000s) and stained following the manufacturer’s guide. Hippocampal neuron images were captured under Z-stack mode using an EVOS FL auto microscope (Life technology) with a 20x object lens for dendritic analysis and a 40x object lens for spines. Dendrites were traced manually using the NeuronJ plugin in ImageJ. Sholl analysis was applied to assess dendrite complexity by measuring the dendritic intersections in concentric circles per 10 μm from the cell soma. Significance was determined by a two-way repeated-measures analysis of variance (RM 2-ANOVA; genotype and circle radius as factors). The analysis of dendritic spine density was performed by calculating spines on the first 50 μm of the apical secondary branch proximate to the cell soma.

Tibias isolated from mice were fixed in 10% neutral buffered formalin then stored in 70% ethanol. Tibia were decalcified in 14% EDTA buffer for 2 weeks at 4 °C, changing the buffer every 2–3 days. Tibia were then embedded in paraffin and sectioned on a microtome to 5 μM sections. Tissues were cleared of paraffin in 2 changes of xylene and hydrated through an alcohol gradient. Slides were then incubated in Bouin’s fixative (Ricca, 1120-16) for 1 h at 55 °C. Slides were rinsed in running dH2O until clear, then stained with hematoxylin (Ricca, 3530-32) for 5 min. To blue the hematoxylin, slides were dipped in 1x TBS for 30 s, then stained in Gomori’s Trichrome solution (Volu-Sol, VXT-032) for 20 min. Slides were then transferred to freshly made 0.5% acetic acid solution for 2 min. The slides were rinsed in dH2O until clear, then dehydrated, cleared in xylene, and mounted with Permount (Fisher, SP15-500). Images were taken at 4x, 10x, and 20x using an EVOS FL Auto microscope (Life Technologies, AMAFD1000). Areas of osteogenesis were quantified as percent of trabecular bone over total marrow area using ImageJ starting 500 μm below the growth plate and continuing for 2 mm.

One thousand single cells were plated into 6-well ultra-low attachment plates (Corning), MammoCult Human Medium (StemCell Technologies) supplemented with Heparin (4 μg/ml, StemCellTechnologies) and Hydrocortisone (0.48 μg/ml, StemCell Technologies) was used. Fresh medium was added every two days. Triptonide was added after culture one week. Mammospheres were counted after 96 h of triptonide treatment.
Images were taken using an EVOS FL Auto microscope (Life Technologies, Carlsbad, CA).