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C2 system

Manufactured by Nikon
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Sourced in Japan, Australia

The C2+ system is a confocal microscope platform from Nikon. It is designed for high-resolution imaging of samples. The system incorporates advanced optics and detection technologies to capture detailed images of biological specimens.

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Lab products found in correlation

Lsm 880, by Zeiss (4 mentions) 4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (4 mentions) Hoechst, by Thermo Fisher Scientific (3 mentions) Phalloidin tritc, by Merck Group (3 mentions) Nis elements software, by Nikon (3 mentions) Cytoflex, by Beckman Coulter (2 mentions) Zen blue software, by Zeiss (2 mentions) μ dishes, by Ibidi (2 mentions) Prolong diamond, by Thermo Fisher Scientific (2 mentions) Triton x 100, by Merck Group (2 mentions) Iba 1, by Abcam (1 mentions) α sma, by Abcam (1 mentions) Elyra 7 lattice sim super resolution microscope, by Zeiss (1 mentions) Plan apochromat 63 na1, by Zeiss (1 mentions) Zen 2, by Zeiss (1 mentions) Elyra ps 1, by Zeiss (1 mentions) Aperio eslide manager, by Leica Microsystems (1 mentions) Aperio at turbo, by Leica Microsystems (1 mentions) Zen black software, by Zeiss (1 mentions) Elyra ps1 system, by Zeiss (1 mentions)

17 protocols using c2 system

1

Localization of Donor Microvesicles in Lung

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MVs were labeled with a DiO Green Fluorescent (Beyotime, China) as per the manufacturer's protocol. Immunofluorescence localization of donor MVs was performed on 10 μm thick cryostat sections on PN9 (48 hours after MV administration). The following primary antibodies were used as markers of alveolar epithelial type I cells (AT1), AT2, vascular endothelial cells, vascular pericytes, total macrophages, and smooth muscle cells: Aquaporin-1 (AQP1, 1 : 200, Abcam), prosurfactant protein C (SP-C, 1 : 100, Abcam), CD31 (1 : 100, Abcam), NG2 (1 : 200, Abcam), Iba-1 (1 : 200, Abcam), and α-smooth muscle actin (α-SMA) (1 : 200, Abcam), respectively. Then, Cy-3 dye-labeled IgG was used as the secondary antibody (Beyotime, China). Fluorescence was observed on Leica laser confocal microscopy (C2+ system, Nikon, Japan), and at least five different visual fields were randomly selected from each sample.
Zhou O., You J., Xu X., Liu J., Qiu H., Hao C., Zou W., Wu W., Fu Z., Tian D, & Zou L. (2022). Microvesicles Derived from Human Umbilical Cord Mesenchymal Stem Cells Enhance Alveolar Type II Cell Proliferation and Attenuate Lung Inflammation in a Rat Model of Bronchopulmonary Dysplasia. Stem Cells International, 2022, 8465294.
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2

Super-Resolution Fluorescence Microscopy

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Fluorescence microscopy was performed using a Zeiss ELYRA PS.1 equipped with an LSM880 (Carl Zeiss, Jena) and a 20×, 63× oil, or 100× oil immersion objective or a C2+ system (Nikon). Super‐resolution images were generated by structured iIlumination microscopy (SIM) using 405, 488, and 561 nm widefield laser illumination. SIM confocal images were processed using the ZEN2.3 software (Carl Zeiss, Jena). For the p65 translocation assay, laser scanning microscopy was performed using the 405, 488, and 561 laser illumination set in individual channels to avoid cross‐talk. For live‐cell experiments, imaging was carried out using an ELYRA 7 Lattice‐SIM Super‐Resolution microscope (Zeiss, Oberkochen, Germany) equipped with a 63× oil objective (Plan‐Apochromat 63×/NA1.4).
Wu Z., Berlemann L.A., Bader V., Sehr D.A., Dawin E., Covallero A., Meschede J., Angersbach L., Showkat C., Michaelis J.B., Münch C., Rieger B., Namgaladze D., Herrera M.G., Fiesel F.C., Springer W., Mendes M., Stepien J., Barkovits K., Marcus K., Sickmann A., Dittmar G., Busch K.B., Riedel D., Brini M., Tatzelt J., Cali T, & Winklhofer K.F. (2022). LUBAC assembles a ubiquitin signaling platform at mitochondria for signal amplification and transport of NF‐κB to the nucleus. The EMBO Journal, 41(24), e112006.
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3

Apoptosis Assay of Glioblastoma Spheroids

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Spheroids were grouped in 8 experimental classes: non-treated controls (w or w/o AMF), cells treated with AbLMNVs (w or w/o AMF), TMZ-AbLMNVS (w or w/o AMF) or free drug (TMZ, w or w/o AMF). Chronic AMF stimulations were carried out with a MagneTherm™ equipment (NanoTherics; 20 mT, 753 Hz), 2 h per day, for 4 days. During AMF exposure, temperature data were collected by using a fiber optic temperature sensor (Osensa). After 4 days, spheroids were collected, washed twice in PBS, treated with trypsin (10 min at 37°C), and dissociated to single cells by pipetting; samples were centrifuged and cells resuspended in annexin V binding buffer (1×) supplemented with 2.5 μM of annexin V-FITC and 1 μg ml-1 of propidium iodide (PI). The staining solution was incubated for 15 min at 37°C protected from light. Fluorescence intensity of the cells stained for annexin V-FITC/PI was evaluated using a Beckman Coulter CytoFLEX (for V-FITC, λex 488 nm; λem 525 ± 40 nm were used; for PI, λex 488 nm; λem 690 ± 50 nm were used). The percentages of early / late apoptotic, necrotic and healthy cell populations were analyzed using the CytoFLEX software and subsequently reported on column graphs. Moreover, after the chronic treatment, spheroids were imaged by transmission light (CLSM; C2 system; Nikon) and their equivalent diameter was plotted on the graph.
Marino A., Camponovo A., Degl'Innocenti A., Bartolucci M., Tapeinos C., Martinelli C., De Pasquale D., Santoro F., Mollo V., Arai S., Suzuki M., Harada Y., Petretto A, & Ciofani G. (2019). Multifunctional temozolomide-loaded lipid superparamagnetic nanovectors: Dual targeting and disintegration of glioblastoma spheroids by synergic chemotherapy and hyperthermia treatment. Nanoscale, 11(44), 21227-21248.
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4

Histological Analysis of Macrophage and αSMA Expression

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Harvested tissues were fixed in 4% paraformaldehyde (PFA) overnight and embedded in paraffin. All specimens were cut to 4‐μm‐thick sections and subjected to hematoxylin and eosin (H&E), ISH, and IHC (Ab information in Appendix Table S5). IHC and quantification of macrophages and αSMA expression were performed as previously described (Mori et al, 2006, 2008, 2014). Observations were made via digital whole slide scanning system (Aperio AT Turbo; Leica Microsystems, Tokyo, Japan) confocal microscopy (C2+ system; Nikon Corp., Tokyo, Japan)]. Aperio eSlide Manager (Leica Microsystems), NIS‐Elements C software version 4.13 (Nikon Corp.), AR software version 4.0 (Nikon Corp.), or IMARIS 7.7.2 (Bitplane, Zurich, Switzerland) were used for data analysis.
de Kerckhove M., Tanaka K., Umehara T., Okamoto M., Kanematsu S., Hayashi H., Yano H., Nishiura S., Tooyama S., Matsubayashi Y., Komatsu T., Park S., Okada Y., Takahashi R., Kawano Y., Hanawa T., Iwasaki K., Nozaki T., Torigoe H., Ikematsu K., Suzuki Y., Tanaka K., Martin P., Shimokawa I, & Mori R. (2018). Targeting miR‐223 in neutrophils enhances the clearance of Staphylococcus aureus in infected wounds. EMBO Molecular Medicine, 10(10), e9024.
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5

Super-resolution microscopy of aSyn aggregates

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Fluorescence microscopy was performed using a Zeiss ELYRA PS.1 system equipped with an LSM880 (Carl Zeiss, Oberkochen) and a 20x/0.8, 63x/1.4 oil or 100x/1.46 oil immersion objective or a C2+ system (Nikon). Super-resolution images were generated by the Structured Illumination (SIM) technology using 405, 488, 561, and 647 nm widefield laser illumination. SIM confocal images were processed using the ZEN Black software (Carl Zeiss, Oberkochen), image data were exported using the ZEN Blue software for further use. For the analysis of stained human brain sections, laser scanning microscopy was performed using the 405, 488, 561, and 647 nm laser illumination set in individual channels to avoid cross-talk. The pinhole was adjusted to generate optical section of 2–5 µm, the acquisition settings were kept constant throughout the experiment. For the analysis of p62 positive aSyn aggregates in human brain sections, a 2 × 2 tile scan using the 20x/0.8 objective acquired and subsequently stitched with ZEN Black software.
Furthmann N., Bader V., Angersbach L., Blusch A., Goel S., Sánchez-Vicente A., Krause L.J., Chaban S.A., Grover P., Trinkaus V.A., van Well E.M., Jaugstetter M., Tschulik K., Damgaard R.B., Saft C., Ellrichmann G., Gold R., Koch A., Englert B., Westenberger A., Klein C., Jungbluth L., Sachse C., Behrends C., Glatzel M., Hartl F.U., Nakamura K., Christine C.W., Huang E.J., Tatzelt J, & Winklhofer K.F. (2023). NEMO reshapes the α-Synuclein aggregate interface and acts as an autophagy adapter by co-condensation with p62. Nature Communications, 14, 8368.
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6

Real-Time Hypochlorite Measurement in Neutrophils

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Measurement of hypochlorite production was detected using APF (Goryo Chemical Inc., Sapporo, Japan) according to the manufacturer's instructions. In brief, 1 drop of tail vein peripheral blood was added to 2 ml of live cell imaging solution (Gibco, Carlsbad, CA, USA), and stained with 10 μM of APF for 30 min at room temperature. APF‐loaded neutrophils were stimulated with 1 μl PMA, and fluorescence images were acquired every 1 min for 60 min using a confocal laser scanning unit microscope (C2+ system, Nikon Corp.) equipped with Plan Apo VC20x (0.75 NA), and the images were processed using IMARIS software (Bitplane).
de Kerckhove M., Tanaka K., Umehara T., Okamoto M., Kanematsu S., Hayashi H., Yano H., Nishiura S., Tooyama S., Matsubayashi Y., Komatsu T., Park S., Okada Y., Takahashi R., Kawano Y., Hanawa T., Iwasaki K., Nozaki T., Torigoe H., Ikematsu K., Suzuki Y., Tanaka K., Martin P., Shimokawa I, & Mori R. (2018). Targeting miR‐223 in neutrophils enhances the clearance of Staphylococcus aureus in infected wounds. EMBO Molecular Medicine, 10(10), e9024.
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7

Cellular Uptake of Fluorescent NLCs

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For confocal microscopy imaging, HDFs were seeded at 1∙104 cells/cm2 in µ-Dishes (35 mm, Ibidi). After 24 h of incubation, 100 μg/ml of DiO-labelled NE_NLCs were added and incubated for a further 24 h. Then, the cells were fixed with 4% paraformaldehyde (PFA, Sigma-Aldrich) at 4°C for 20 min, and rinsed three times with PBS (Sigma-Aldrich). Thereafter, cells were treated with Hoechst (1:1,000 v/v, Invitrogen) and TRITC-phalloidin (1:200 v/v, Sigma-Aldrich) at 37°C for 45 min for nuclei and f-actin staining, respectively. Finally, 2D images and 3D rendering have been acquired with a confocal fluorescence microscope (C2 system Nikon).
For flow cytometry analysis, HDFs (1∙104 cells/cm2) were seeded in a 24-well plate and incubated for 24 h. Then, they were treated with 100 μg/ml of DiO-labelled NE_NLCs. After a further 24 h of incubation, the cells were washed and centrifuged at 610 g for 5 min. The pellets were resuspended in PBS and analyzed by flow cytometry (Beckman Coulter CytoFLEX; λex = 488 nm; λem = 530 nm).
Emanet M., Şen Ö., Pignatelli F., Lavarello C., Petretto A, & Ciofani G. (2022). Hazelnut extract-loaded nanostructured lipid carriers and evaluation of their antioxidant properties. Frontiers in Bioengineering and Biotechnology, 10, 953867.
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8

Migration Assay of RAW264 Cells

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Migration assays were performed using 24 well transwell plates with 8.0 μm pore size inserts. RAW264 cells were cultured in 24 well plates at a density of 4 × 105 cells per well. To identify the migrated cells, RAW264 cells on the upper side were labeled with green fluorescence using the CellTrace CFSE Cell Proliferation Kit (C32554; Thermo Fisher Scientific) according to the manufacturer’s instructions. Carboxyfluorescein succinimidyl ester (CFSE)-labeled cells were seeded into the upper chamber. GTS-21 (100 μM) or α-bungarotoxin (#203980, Sigma-Aldrich) (1 μg ml−1) was added to a medium containing LPS (100 ng ml−1) or PBS as a control, and the number of migrated cells was evaluated after 4 or 24 h. The same treatment medium was then added to the upper and lower chambers. Migrated cells were photographed using a confocal microscopy (C2+ system; Nikon Corporation, Tokyo, Japan), and the images were visualized using IMARIS software (Bitplane, Zurich, Switzerland). The number of CFSE-labeled migrated cells were counted in every three random rows in the field, and the mean value for each group was calculated.
Nakamura Y., Matsumoto H., Wu C.H., Fukaya D., Uni R., Hirakawa Y., Katagiri M., Yamada S., Ko T., Nomura S., Wada Y., Komuro I., Nangaku M., Inagi R, & Inoue T. (2023). Alpha 7 nicotinic acetylcholine receptors signaling boosts cell-cell interactions in macrophages effecting anti-inflammatory and organ protection. Communications Biology, 6, 666.
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9

Cellular Uptake of Functionalized Nanoparticles

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Cells were seeded at 2·104 cells/cm2 in μ-Dishes (35 mm; Ibidi) coated with Matrigel (10 mg/mL) for confocal microscopy imaging. After 24 h, cultures were incubated with 500 μg/mL of DiO-labelled PNPs, Nut-PNPs, ApoE-PNPs, or ApoE-Nut-PNPs for further 24 and 72 h. Then, the cells were fixed using 4% paraformaldehyde (PFA; Sigma-Aldrich) at 4°C for 20 min. Next, they were rinsed three times with PBS. hCMEC/D3 cells were incubated with TRITC-phalloidin (1:200 v/v; Sigma-Aldrich) and Hoechst (1:1000 v/v; Invitrogen) at 37°C for 45 min for the imaging of nuclei and f-actin, respectively. Finally, a confocal fluorescence microscope (C2 system Nikon) was used to acquire 2D images and 3D rendering.
Şen Ö., Marino A., Pucci C, & Ciofani G. (2021). Modulation of anti-angiogenic activity using ultrasound-activated nutlin-loaded piezoelectric nanovectors. Materials Today Bio, 13, 100196.
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10

Cellular Uptake of Nanoparticles in Brain Endothelial Cells

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Cells were seeded at 2·104 ​cells/cm2 in μ-Dishes (35 ​mm; Ibidi) coated with Matrigel (10 ​mg/mL) for confocal microscopy imaging. After 24 ​h, cultures were incubated with 500 ​μg/mL of DiO-labelled PNPs, Nut-PNPs, ApoE-PNPs, or ApoE-Nut-PNPs for further 24 and 72 ​h. Then, the cells were fixed using 4% paraformaldehyde (PFA; Sigma-Aldrich) at 4°C for 20 ​min. Next, they were rinsed three times with PBS. hCMEC/D3 cells were incubated with TRITC-phalloidin (1:200 v/v; Sigma-Aldrich) and Hoechst (1:1000 v/v; Invitrogen) at 37°C for 45 ​min for the imaging of nuclei and f-actin, respectively. Finally, a confocal fluorescence microscope (C2 system Nikon) was used to acquire 2D images and 3D rendering.
Şen Ö., Marino A., Pucci C, & Ciofani G. (2021). Modulation of anti-angiogenic activity using ultrasound-activated nutlin-loaded piezoelectric nanovectors. Materials Today Bio, 13, 100196.
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