Dmi6000 cs microscope

Chemotaxis assay with a µ-Slide Chemotaxis (IBIDI) was performed according to the manufacturer’s manual (Application Note 17 and 23), using conditioned medium from SIPS HDFs and PBMCs freshly isolated from human blood by Ficoll gradient centrifugation. Life cell imaging was performed using a DMI6000 CS microscope (Leica) equipped with a heated CO₂ chamber (OKOLAB) and an automated stage. During 12 h, one image/2.5 min was taken. For each condition 30 cells were tracked using the manual tracking plugin and analysed with the chemotaxis and migration tool plugin (IBIDI) in ImageJ. SIPS 1201 HDFs were pre-treated for 4 days with 1201 prior to the experiment. During the conditioning of the medium and the subsequent chemotaxis assay, 1201 was not added to the medium.

Cell densities were evaluated as previously described (Neher et al., 2011b (link)). In brief, cell densities were assessed in live cultures after staining with the nuclear dyes Hoechst 33342 (5 μg/mL) and propidium iodide (1 μg/mL); Alexa-488–tagged isolectin-B4 (1 μg/mL) was used to identify microglia. Healthy and apoptotic (chromatin-condensed) neurons were recognized by their distinct nuclear morphology, whereas propidium-iodide positive cells (indicating membrane permeabilization) were scored as necrotic. Four microscopic fields/well (between 150 and 200 neurons per field in control wells) in 2 wells/condition were quantified for a single experiment. Experiments performed with cultures from at least three independent culture preparations were used for statistical analysis. For tetramethylrhodamine methyl ester (TMRM) staining, cells were incubated with TMRM (3 nM) for 30 min at 37°C, 5% CO₂. Cell nuclei were counterstained with Hoechst 33342 (10 μg/mL). Total cell densities and TMRM-positive cell numbers were evaluated using a Leica DMI6000 CS microscope.

Leaf discs excised from N. benthamiana were mounted on microscope slides in water and the subcellular localization of xFP-tagged proteins was analyzed using a Leica TCS SP5 confocal unit attached to a Leica DMI6000 CS microscope. GFP and YFP were excited at 488 nm and collected at 500–525 nm and 525–540 nm, respectively. RFP was excited at 561 nm and collected at 580–610 nm. The gain setting of the confocal unit was adjusted to just below the threshold for saturation of the signal. Images were acquired and analyzed using LAS AF software (Leica).

Images were acquired by Leica LAS AF 1.8.2 software with either an inverted Leica SP5 confocal system using a Leica DMI6000CS microscope or an upright Leica SP5 2 confocal system using a Leica DM 6,000 CFS microscope. Using the inverted microscope, images were acquired using a × 10 Leica Plan Apochromat objective with 0.40 numerical aperture for quantification and a × 20 Leica Plan Apochromat objective with 0.70 numerical aperture. Using the upright microscope, images were acquired using an HCX APO L20x objective with a 1.0 numerical aperture for still images and subsequent movies. Imaging of calvarium ranged from 60 to 100 μm. CFP (excitation 458 nm and emission 463–500 nm), GFP (excitation 488 nm, emission 493–556 nm) and DsRed2 (excitation 561 nm, emission 566–650 nm) were excited with an Argon/2 (458, 477, 488, 496 and 514 nm) and Diode pumped solid-state (561 nm) laser, respectively. The power used for dsRed visualization was 8–12% of the appropriate laser. Images were continuously captured in 1,024 × 1,024 or 1,024 × 512 format, with line averaging of 4 (∼10 or 5 s per scan, respectively) for up to 8 h. Multicolour imaging for CFP and GFP were captured sequentially.

Murine microglial BV2 cell line (passage <30) was cultured as previously described22 (link). Primary mixed neuronal/glial cultures were prepared from cerebella of postnatal day 5 to 7 Wistar rats (male and female). Neurons were plated at a density of 5 × 10⁵ cells per well in a 24-well plate and medium was changed once 24 hours after the cells were seeded. The cell cultures were left for one week before treatment. The approximate composition of the mixed cultures is 85 ± 5% neurons, 7 ± 3% astrocytes, and 5 ± 3% microglia. For live-cell counts, cultures were incubated with the nuclear stains Hoechst 33342 (5 μg/ml) and propidium iodide (1 μg/ml); Alexa 488-tagged isolectin-B4 (1 μg/ml) was used to distinguish microglia. Healthy and apoptotic (chromatin-condensed) neurons were recognized by their distinct nuclear morphology, whereas propidium iodide-positive cells (indicating membrane permeabilization) were identified as necrotic. Cell densities were evaluated using a Leica DMI6000 CS microscope. Four microscopy fields per well were quantified (around 150–200 neurons per field in control conditions), and experiments were performed with three independent culture preparations.