Af6000

Manufactured by Leica

Sourced in Germany, Austria, Switzerland, Japan, United States

The AF6000 is a high-precision autofocus module designed for microscopy applications. It features fast and accurate autofocus capabilities to support a wide range of imaging techniques.

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Lab products found in correlation

Dfc360 fx camera, by Leica (8 mentions) 4 6 diamidino 2 phenylindole (dapi), by Merck Group (7 mentions) Dmi6000b, by Leica (5 mentions) Las af software, by Leica (5 mentions) Dmi6000, by Leica (5 mentions) Alexa fluor 488, by Thermo Fisher Scientific (4 mentions) Dmi6000 b with imc, by Leica (4 mentions) Nicolet is50, by Thermo Fisher Scientific (3 mentions) Dmi4000b fluorescence microscope system, by Leica (3 mentions) Fw4000, by Leica (2 mentions) Fluorescent mounting media, by Vector Laboratories (2 mentions) Lsm 700 confocal microscope, by Leica (2 mentions) Microwell 96 well glass bottom plates, by Thermo Fisher Scientific (2 mentions) Alexa fluor secondary antibody, by Thermo Fisher Scientific (2 mentions) Dfc350fx, by Leica (2 mentions) 4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions) Propidium iodide, by Thermo Fisher Scientific (2 mentions) Dmi6000b microscope, by Leica (2 mentions) Escalab 250xi, by Thermo Fisher Scientific (2 mentions) Prolong gold antifade reagent, by Thermo Fisher Scientific (2 mentions)

Close spelling variants of this product

AF6000 imaging system (2 citations) AF6000 Leica Software v3.1.0 (3 citations) Leica Application Suite AF6000 Module Systems (2 citations) DMI6000-AF6000 Leica microscopic imaging system (2 citations) AF6000 Live Cell Imaging System (2 citations) AF6000 LX Widefield Multidimensional Microscopy System (3 citations) AF6000 Time Lapse microscope (4 citations) AF6000-DMI6B (2 citations) AF6000 LX system microscope (2 citations) AF6000 cell station (8 citations)

157 protocols using af6000

Quantitative Microscopy of Cell Morphology

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For co-culture experiments images were taken every hour at different magnifications with an inverted fluorescence microscope (Leica AF6000). Cancer cell elongation was quantified manually by scoring individual cells using Image J “Cell Counter” plugin on taken images at 48 h co-culture. Elongated cell were defined as single cell with a minimal length-width rapport of 2:1, as well as at least one sharp-ending extremity. Cancer cells motility was quantified based on single cell track follow on 24 h microscope movies using Image J “Manual Tracking” and “Chemotaxis Tool” plugins.
Imaging of 2D and 3D spheroids assay were performed with 5× and 40× objectives with Leica AF6000 inverted fluorescence microscope. Invasion was quantified using Photoshop CS6 (Adobe) by calculating the invaded area on the 4 days image, normalized on initial spheroid size.
Western Blot images were quantified for protein level using Image J “Gel Analyze Tool” plugin.
For immunostaining images were taken using a DeltaVision Elite Microscope (GE Healthcare) with 63× and 100× objectives, followed by deconvolution treatment.
For immunohistochemistry images, slides were scanned using a NanoZoomer 2.0 HT (Hamamatsu) and images were extracted using NDP.view2 analysis software (Hamamatsu).

Knuchel S., Anderle P., Werfelli P., Diamantis E, & Rüegg C. (2015). Fibroblast surface-associated FGF-2 promotes contact-dependent colorectal cancer cell migration and invasion through FGFR-SRC signaling and integrin αvβ5-mediated adhesion. Oncotarget, 6(16), 14300-14317.

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Live-Cell Imaging of Cell Migration

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To examine cell migration, live-cell imaging was performed on a Leica AF6000 inverted microscope at 37 °C under 5% CO₂. H1299 cells were seeded in six-well plates and grown to 100% confluence. A ‘wound’ was created using pipette tips. Cells were washed with 1 × PBS and maintained in DMEM with 1% FBS. The cell images at exact same place were recorded every 30 min using a time-lapse microscopy (Leica AF6000).

Lv T., Wu X., Sun L., Hu Q., Wan Y., Wang L., Zhao Z., Tu X, & Xiao Z.X. (2017). p53-R273H upregulates neuropilin-2 to promote cell mobility and tumor metastasis. Cell Death & Disease, 8(8), e2995-.

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Colony Formation Assay for Adriamycin-Resistant K562 Cells

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Following treatment of the K562 cells (concentrations of ADM used were 0.1, 0.2, 0.4, 0.6 and 0.8 µmol/ml) and K562/ADM cells (concentrations of ADM used were 1, 2, 4, 6 and 8 µmol/ml), and culture medium removal, the adherent cells were then rinsed with phosphate-buffered saline (PBS) once, and detached with 0.25% trypsin [without ethylene diamine tetraacetic acid (EDTA)]. The cells were then triturated into a single cell suspension and suspended in complete medium, and 200 cells/well were inoculated into a 6-well plate. The plate was shaken gently, so that the cells were evenly dispersed and were cultured for 1-2 weeks. When cell clones (>50) were visible to the naked eye, following cultivation termination and culture medium removal, the cells were washed 3 times with PBS and fixed 10 min with anhydrous methanol. Subsequently, the cells were stained with Giemsa solution (Sigma-Aldrich; Merck KGaA) for 15 min, and the dying solution was washed away slowly with running water and the cells were dried and photographed under a microscope (Leica, AF6000, Leica Microsystems GmbH). The experiment was repeated 3 times. Under the microscope (low power microscope) (AF6000, Leica Microsystems GmbH), the colonies with >10 cells were counted. The data are expressed as the means ± standard deviation.

Wuxiao Z., Wang H., Su Q., Zhou H., Hu M., Tao S., Xu L., Chen Y, & Hao X. (2020). MicroRNA-145 promotes the apoptosis of leukemic stem cells and enhances drug-resistant K562/ADM cell sensitivity to adriamycin via the regulation of ABCE1. International Journal of Molecular Medicine, 46(4), 1289-1300.

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Immunohistochemical Analysis of HMGB1 in Myocardial Infarction

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Hearts fixed in 4% paraformaldehyde were embedded in paraffin and then cut into 5 μm sections. Sections were incubated with an anti-HMGB1 rat polyclonal primary antibody (1:200 dilution, 60 min at room temperature; Abcam, USA) and a secondary antibody (1:100 dilution, 60 min at room temperature; Zhongshan Biotechnology, China). The sections were incubated in HRP-streptavidin (1:100; Zhongshan Biotechnology, China) for 30 min at 37°C, and the color reaction was visualized with diaminobenzidine (DAB; ZSGB-BIO, Beijing, China). For each section, five fields were randomly selected from the infarcted area of the left ventricle and photographed with a digital camera (AF6000, Leica, Germany). The mean density of HMGB1 expression in the myocardium was analyzed using Image J.

Zhang J., Xia F., Zhao H., Peng K., Liu H., Meng X., Chen C, & Ji F. (2019). Dexmedetomidine-induced cardioprotection is mediated by inhibition of high mobility group box-1 and the cholinergic anti-inflammatory pathway in myocardial ischemia-reperfusion injury. PLoS ONE, 14(7), e0218726.

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Adenoviral Transfection of Rat Cardiomyocytes

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Recombinant adenovirus with β₂-AR or EGFP genes were used to transfect myocardial cells of rats, at a multiplicity of infection of 100. At 48 h after transfection, the myocardial cells were removed from the incubator, observed and photographed under a fluorescence microscope (AF6000, Leica (Wetzlar, Germany). Five views were randomly selected and transfection efficiencies, as the number/total number of stab cells with the expression of green fluorescence under the same view, were calculated, respectively.

Lin Y., Zheng C., Liu Y., Wang L, & Gong H. (2016). Effect of adenovirus mediated β2-AR overexpression on IL-10 level secreted by cardiomyocytes of heart failure rats. Experimental and Therapeutic Medicine, 12(3), 1349-1354.

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Immunofluorescence Assay for Virus Isolation

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To confirm that the virus had been successfully isolated and had infected the cells, IFA was performed. Briefly, after the F81 cells in the six-well plates were inoculated and cultured with the supernatants containing the virus for 2 days, they were fixed for 30 min at 4°C with pre-cooled 70% ethanol. Then, the cells were incubated in 1% BSA for 1 h at 37°C to block non-specific protein–protein interactions. Subsequently, the cells were incubated with mouse anti-CPV-2 monoclonal antibodies cross-reacting with FPV, MEV, and RDPV (Qianxun Biological Company, catalog. no: Ab-015) using the dilution of 1:50 overnight at 4°C. The secondary antibody used was a FITC-goat anti-mouse IgG antibody (Jackson ImmunoResearch Laboratories, Inc., catalog. no: 115–035\u2013003) with a dilution of 1:200 and incubated for 1 h at 37°C. Finally, a fluorescent strain (4′,6-diamidino-2-phenylindole, DAPI) for 5 min at room temperature was used to stain the cell nucleus. The cells were observed under a fluorescence microscope with a 10 × objective (Leica AF6000, Germany).

Zhao J., Zhang H., Zhang L., Zhang Q., Zhou N., Du T., Zhao Q., Zhou E.M., Du Y, & Sun Y. (2022). Isolation and Genetic Characterization of Parvoviruses From Dogs, Cats, Minks, and Raccoon Dogs in the Eastern Region of Shandong Province, China. Frontiers in Microbiology, 13, 862352.

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Viability Staining of HPAECs

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HPAECs were cultured on coverslips until the cell confluence reached 80%. The cells were treated with different agents according to the different experimental groups. Afterward, the cells were stained with 6 μL of Hoechst 33342 solution and 6 μL of PI (propidium iodide) at 4 °C in the dark for 20 min. Images were captured with a living cell workstation (AF6000; Leica, Germany).

Wang X., Li Q., He S., Bai J., Ma C., Zhang L., Guan X., Yuan H., Li Y., Zhu X., Mei J., Gao F, & Zhu D. (2022). LncRNA FENDRR with m6A RNA methylation regulates hypoxia-induced pulmonary artery endothelial cell pyroptosis by mediating DRP1 DNA methylation. Molecular Medicine, 28, 126.

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Immunohistochemical Analysis of sFGFR2IIIc Mutant

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Tissues were fixed in 4% paraformaldehyde in PBS for 24 h at 4°C and were then dehydrated in a graded series of ethanol, embedded in paraffin, and sectioned at 7-µm intervals. After the sections were dewaxed and rehydrated, they were stained with hematoxylin and eosin (Wako). To detect whether sFGFR2IIIc^S252W was applied, immunohistochemical staining was performed using polyclonal anti-FLAG (primary) and anti-rabbit IgG HRP-conjugated (secondary) antibodies. Immunoreactivity was subsequently visualized using a VECTASTAIN ABC Kit (Vector Laboratories, Peterborough, UK) according to the manufacturer's instructions. Sections were counterstained with Nuclear Fast Red (Vector Laboratories). BrdU incorporation was also examined as described above. For immunodetection of incorporated BrdU, cells were incubated with fluorescein isothiocyanate-conjugated anti-BrdU monoclonal antibodies for 45 min at 37°C. Cell nuclei were stained with 5 mg/mL 4′,6-diamidino-2-phenylindole (DAPI; Sigma) in PBS prior to mounting slides. Fluorescence was visualized using a fluorescence microscope (AF6000; Leica, Solms, Germany). The frequency of S-phase cells was calculated as the ratio of BrdU-positive nuclei to total DAPI-stained nuclei.

Yokota M., Kobayashi Y., Morita J., Suzuki H., Hashimoto Y., Sasaki Y., Akiyoshi K, & Moriyama K. (2014). Therapeutic Effect of Nanogel-Based Delivery of Soluble FGFR2 with S252W Mutation on Craniosynostosis. PLoS ONE, 9(7), e101693.

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Live-cell Imaging of PRRSV Infection

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MARC-145 cells were grown on a 35 mm cell culture dish to a density of 50–70% confluence and then infected with rSD16/TRS2-DsRed (MOI: 0.1) of different passages. After 1 h post-infection (hpi), the medium was removed and replaced with 2 mL of pre-warmed DMEM containing 3% FBS. The dish was placed at 37°C with 5% CO₂. The fluorescence of different passages of rSD16/TRS2-DsRed was captured by Leica microscope at 36 hpi. In live-cell imaging, MARC-145 cells were infected with rSD16/TRS2/DsRed at an MOI of 0.01 in a 35 mm cell culture dish. At 1 hpi, the medium was removed and replaced with 2 ml of pre-warmed DMEM containing 3% FBS. The dish was placed in the 37°C observation chamber containing 5% CO₂ (Leica CTR-Controller 3700, Wetzlar, Germany). DsRed signals and phase-contrast images were captured with the time interval of 1 min for 3 h by a live-cell station (Leica AF6000, Wetzlar, Germany). Subsequently, images were processed into a movie of 10 frames s-1 using QuickTime Pro.

Li N., Zhang Y., Yao L., Shi Y., Zhao Q., Huang B, & Sun Y. (2022). A Recombinant Porcine Reproductive and Respiratory Syndrome Virus Stably Expressing DsRed Protein Based on Bacterial Artificial Chromosome System. Frontiers in Microbiology, 13, 839845.

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Immunofluorescence Assay for IGF2BP1

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The DEE cells were rinsed with PBS, fixed with 0.2% polyoxymethylene for 10 min at room temperature, and then incubated with mouse anti-IGF2BP1 mAb (Abcam, 1:3000) for 1 h at 37 °C. After reaction with fluoresceine isothiocyanate (FITC)-conjugated goat anti-mouse antibody (Abcam, 1:3000), the samples were washed three times with PBS and inoculated with 500 μL 4′,6-diamidino-2-phenylindole (DAPI; Merck, Germany) for 10 min. The slips were then visualized by laser scanning confocal microscopy (Leica AF6000).

Chen J., Zhang R., Lan J., Lin S., Li P., Gao J., Wang Y., Xie Z.J., Li F.C, & Jiang S.J. (2019). IGF2BP1 Significantly Enhances Translation Efficiency of Duck Hepatitis A Virus Type 1 without Affecting Viral Replication. Biomolecules, 9(10), 594.

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