Axiovision 40v 4

Striatal GFP neurons were examined on sections using a Zeiss Axio Observer Z1 fluorescent microscope equipped with a Zeiss Colibri LED illumination system and a Zeiss AxioCam MRc camera (Zeiss, Oberkochen, Germany), with a 20× objective. We used 9 × 9 tiles with a 10% overlap to create panoramic images. Mosaic stitching was carried out using AxioVision 40 V 4.8.2.0 software (Zeiss, Oberkochen, Germany).
The diameter and area of the neuron cell bodies were determined on sections with the AxioVision 40 V 4.8.2.0 software using the line and outline tools, respectively. This analysis was performed on cells with a visible nucleus and axonal hillock. The neuron size was determined by drawing a line from the axonal hillock to the opposite edge of the cell.
For microscopy of living neurons stained with DRAQ5 and GFP, the sorted suspension was placed on a coverslip and covered with a round coverslip, 15 mm in diameter. The resulting preparations were studied under a Zeiss Axio Observer Z1 fluorescent microscope. Images were acquired at a 40× objective and analyzed using AxioVision 40 V 4.8.2.0 software. Living neuron microscopy was performed over the entire area of the coverslip, 176.7 mm², analyzing images of all sorted cells.

EVs were purified from U1 and U937 cell culture and separated into 10 K and 100 K EVs. A 1.5 μL fluorescent label of BODIPY™ 493/503 (Cat. # D3922; Invitrogen™) was mixed with 50 μL EVs and incubated for 30 min at 37 °C. Any unbound BODIPY was filtered out using a Pharmacia G-50 spin column (1 mL bed volume in PBS buffer; 2000 rpm/2 min; Sorval RT6000D), yielding 30 μL of labeled EVs. In biological triplicate, five microliters of labeled EVs were added onto MDMs (50,000 cells on each coverslip in 200 μL at cell: EV ratios 1:10,000). Treated cells were analyzed with confocal microscopy at the UNMC core facility. The prolong gold antifade mounted slides were imaged in Zeiss Observer.Z1 microscope equipped with a monochromatic Axiocam MRm camera using Axiovision 40 v.4.8.0.0 software (Carl Zeiss, Oberkochen, Germany). The red, green, and blue colors were assigned to Alexa Fluor 568, KC57-FITC, and DAPI, respectively.