Ix81 fluorescence microscope

Manufactured by Olympus

Sourced in Japan, Germany, United States, Poland

The IX81 is a fluorescence microscope designed for biological research. It features a motorized stage and filter wheel for automated imaging. The IX81 is capable of fluorescence observation and imaging.

Automatically generated - may contain errors

Lab products found in correlation

4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (7 mentions) Hoechst 33342, by Thermo Fisher Scientific (6 mentions) Triton x 100, by Merck Group (4 mentions) Xm10 camera, by Olympus (4 mentions) Bovine serum albumin (bsa), by Merck Group (3 mentions) Cellp software, by Olympus (3 mentions) Prolong gold, by Thermo Fisher Scientific (3 mentions) Paraformaldehyde (pfa), by Merck Group (3 mentions) Streptavidin alexa 488, by Thermo Fisher Scientific (3 mentions) Scanr version 2, by Olympus (2 mentions) Scanr, by Olympus (2 mentions) Paraformaldehyde (pfa), by Avantor (2 mentions) 4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions) Mineral oil, by Trinity Biotech (2 mentions) Ccd camera, by Hamamatsu Photonics (2 mentions) Trap staining solution, by Fujifilm (2 mentions) Infinite m1000 pro, by Tecan (2 mentions) Metamorph software, by Universal Imaging (2 mentions) Fv 1000 confocal set up, by Olympus (2 mentions) Alexa fluor dye labeled secondary antibody, by Thermo Fisher Scientific (2 mentions)

148 protocols using ix81 fluorescence microscope

Quantifying Cellular ROS Production

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Total ROS production was evaluated in PMNs by two methods. Cells were incubated (30 min) with the fluorescent probe (5 x 10⁻⁶ mol/L) 2’,7’-dichlorodihydrofluorescein diacetate (DCFH-DA) [24 (link)]. First, ROS production was assessed by fluorimetry using a Synergy Mx plate reader (BioTek Instruments, Winooski, VT). Second, it was assessed using a fluorescence microscope (IX81, Olympus, Hamburg, Germany) coupled with the static cytometry software ‘ScanR’ version 2.03.2 (Olympus). For static cytometry, PMNs from each subject were seeded in triplicate in 48-well plates and 16 images per well were recorded and analyzed [24 (link)].
The fluorescent probe Mitosox Red (5μM) was employed to assess mitochondrial ROS production. Leukocytes were seeded in 48-well plates and incubated for 30 min with the respective fluorochrome and washed with HBSS. 16 images per well were recorded with an IX81 Olympus fluorescence microscope (Olympus, Hamburg, Germany), and the static cytometry software ‘ScanR’ version 2.03.2 (Olympus) was used to quantify fluorescence individually (per cell). Fluorescent probes were purchased from Invitrogen (Life Technologies, Barcelona, Spain).

Victor V.M., Rovira-Llopis S., Bañuls C., Diaz-Morales N., Martinez de Marañon A., Rios-Navarro C., Alvarez A., Gomez M., Rocha M, & Hernández-Mijares A. (2016). Insulin Resistance in PCOS Patients Enhances Oxidative Stress and Leukocyte Adhesion: Role of Myeloperoxidase. PLoS ONE, 11(3), e0151960.

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Neurite Length Assessment in SH-SY5Y Cells

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For neurite lengths assessment in SH-SY5Y neuron-like cells, multiple representative fields of cells morphology stained with MAP-2 labeling were photographed with an IX-81 OLYMPUS fluorescence microscope (OLYMPUS, Germany) fitted to a DP-711 digital camera (OLYMPUS, Germany). Captured images were labeled with a scale according to the correspondent microscope magnification (40x). The images scale was used to convert pixels units into micrometers (μm), using for this purpose the NIH Image J (Rasband WS, Image J, NIH), version 1.41. In addition, the channels were extracted to grey scale and the length of 5 to 10 neurites per field was traced and measured, thereafter, from the distal end of neuron growth-cone, using the neurite tracer plugin of NIH Image [48 (link), 49 (link)].

Pires A.O., Neves-Carvalho A., Sousa N, & Salgado A.J. (2014). The Secretome of Bone Marrow and Wharton Jelly Derived Mesenchymal Stem Cells Induces Differentiation and Neurite Outgrowth in SH-SY5Y Cells. Stem Cells International, 2014, 438352.

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Superoxide Production Quantification

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Determination of superoxide production was assessed by fluorometry using an IX81 Olympus fluorescence microscope coupled with the static cytometry software ScanR version 2.03.2 (Olympus, Hamburg, Germany). Leukocytes were seeded in a 48-well plaque and incubated for 30 min at 37 °C with a Dihydroethidium (DHE) probe for intracellular superoxide determination and with Hoechst 33342 to visualize cell nuclei. Both fluorescent dyes were purchased from Life Technologies (Thermo Fisher Scientific, Waltham, MA, USA).

Abad-Jiménez Z., López-Domènech S., Gómez-Abril S.Á., Periañez-Gómez D., de Marañón A.M., Bañuls C., Morillas C., Víctor V.M, & Rocha M. (2020). Effect of Roux-en-Y Bariatric Bypass Surgery on Subclinical Atherosclerosis and Oxidative Stress Markers in Leukocytes of Obese Patients: A One-Year Follow-Up Study. Antioxidants, 9(8), 734.

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Intracellular Growth Assay of Legionella pneumophila

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For infection experiments, L. pneumophila strains were grown to the post-exponential phase as measured by optical density of the culture (OD₆₀₀ = 3.3–3.8) and judged by an increase in bacterial motility. For the L. pneumophila intracellular growth assay, we prepared bone marrow-derived macrophages from femurs of A/J mice using an established protocol [31 (link)] with 30% L-cell-conditioned medium in RPMI1640 containing 10% FBS. 4.0 × 10⁵ macrophages were seeded on glass coverslips in 24-well plates. The cells were then infected with the indicated L. pneumophila strains expressing GFP from pAM239 [32 (link)] at an MOI of 0.5 and grown at 37 °C in the presence of 5% CO₂. After 2 h, the infection was synchronized by washing the monolayers three times with warmed, sterile PBS. At 14 hpi, cells attached to coverslips were washed 3 times with PBS and fixed using 4% paraformaldehyde at room temperature for 20 min. The coverslips were mounted and imaged using an IX-81 Olympus fluorescence microscope and the bacteria per vacuole were enumerated.

McCloskey A., Perri K., Chen T., Han A, & Luo Z.Q. (2021). The metaeffector MesI regulates the activity of the Legionella effector SidI through direct proteine–protein interactions. Microbes and infection, 23(4-5), 104794.

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Superoxide and Mitochondrial Membrane Potential Assay

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The determination of superoxide production and mitochondrial membrane potential was assessed by static fluorometry using an IX81 Olympus fluorescence microscope coupled with the static cytometry software ScanR v2.03.2 (Olympus, Hamburg, Germany). In brief, 1.5 × 10⁵ PMN/wells were seeded in a 48-well plaque and incubated for 30 min at 37 °C with Dihydroethidium (DHE) and tetramethylrhodamine methyl ester (TMRM) probes for intracellular superoxide and mitochondrial membrane potential determination, respectively. Hoechst 33,342 was used to visualise cell nuclei. All fluorescent dyes were purchased from Life Technologies (Thermo Fisher Scientific, Waltham, MA, USA).

Abad-Jiménez Z., Vezza T., López-Domènech S., Fernández-Reyes M., Canet F., Morillas C., Gómez-Abril S.Á., Bañuls C., Víctor V.M, & Rocha M. (2022). Impact of Roux-en-Y Gastric Bypass on Mitochondrial Biogenesis and Dynamics in Leukocytes of Obese Women. Antioxidants, 11(7), 1302.

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Multiparametric analysis of leukocyte function

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Total ROS production, GSH content and membrane potential were determined using the fluorescent probes 2′, 7′-dichlorodihydrofluorescein diacetate (DCFH-DA, 5 μM) 5-chloromethylfluorescein diacetate (CMFDA, 2.5 μM) and tetramethylrhodamine methylester (TMRM, 5 μM), respectively. The leukocytes were seeded in 48-well plates and incubated with the probes for 30 min. Next, the wells were washed with HBSS and data was acquired with an IX81 Olympus fluorescence microscope (Olympus, Hamburg, Germany) and static cytometry software ‘ScanR’ version 2.03.2 (Olympus). Single cell fluorescence was measured and quantified. All probes were purchased from Invitrogen (Life Technologies, Barcelona, Spain). In order to assess Oxygen consumption, leukocytes were resuspended at a density of 5 × 10⁶ cells/mL in HBSS and placed in a gastight chamber coupled to a Clark-Type O₂ electrode (Rank Brothers). In order to check whether O₂ consumption was mainly mitochondrial, Sodium cyanide (10^− 3 M) was employed.

Bañuls C., de Marañon A.M., Veses S., Castro-Vega I., López-Domènech S., Salom-Vendrell C., Orden S., Álvarez Á., Rocha M., Víctor V.M, & Hernández-Mijares A. (2019). Malnutrition impairs mitochondrial function and leukocyte activation. Nutrition Journal, 18, 89.

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Mitochondrial ROS and Inflammatory Biomarkers

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To detect mitochondrial ROS, leukocytes were seeded in a 48-well plate (1.5 × 10⁵ cells/well) and incubated for 30 min with MitoSOX dye (Life Technologies, Thermo Fisher Scientific, Waltham, MA, USA) prepared at 5 μM in HBSS. The nuclei were visualized using the nuclear stain Hoechst 33342. The fluorescence signal was detected with an IX81 Olympus fluorescence microscope and analyzed with ScanR software version 2.03.2 (Olympus, Shinjuku, Japan).
Serum levels of interleukin 6 (IL6), IL1β, interleukin 18 (IL18), TNFα and myeloperoxidase (MPO) were measured with a Luminex^® 200 analyzer system (Austin, TX, USA) following the Milliplex^® MAP Kit manufacturer’s procedure (Millipore Corporation, Billerica, MA, USA). The intra-serial and inter-serial variation coefficients were <5.0% and <20.0%, respectively.

Abad-Jiménez Z., López-Domènech S., Díaz-Rúa R., Iannantuoni F., Gómez-Abril S.Á., Periañez-Gómez D., Morillas C., Víctor V.M, & Rocha M. (2020). Systemic Oxidative Stress and Visceral Adipose Tissue Mediators of NLRP3 Inflammasome and Autophagy Are Reduced in Obese Type 2 Diabetic Patients Treated with Metformin. Antioxidants, 9(9), 892.

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Multiparametric Analysis of Cell Biology

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Cell proliferation/survival, mitochondrial membrane potential, mass and superoxide production, ER signal, lysosome signal and intracellular lipid accumulation were analyzed using specific fluorochromes with an IX81 Olympus fluorescence microscope with “ScanR” static cytometry software version 2.03.2 (Olympus, Hamburg, Germany). Details are provided as Supplementary Material.

Alegre F., Martí-Rodrigo A., Polo M., Ortiz-Masiá D., Bañuls C., Pinti M., Álvarez Á., Apostolova N., Esplugues J.V, & Blas-García A. (2022). Macrophages Modulate Hepatic Injury Involving NLRP3 Inflammasome: The Example of Efavirenz. Biomedicines, 10(1), 109.

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Apoptosis assessment using Hoechst-PI staining

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After treatment with GSK343 +/-TRAIL for 24 hours apoptosis induction was assessed by Hoechst 33343 and propidium Iodide (PI) stain (Sigma-Aldrich, Dorset, England).
Cells were incubated with 10µg/ml Hoechst 33342 and 10µg/ml PI at 37°C for 30 minutes examined using IX81 Olympus fluorescence microscope, using Cell F software for image capture. Images were captured using a UC30 colour camera and triple filter for DAPI/FITC/TRITC. Images containing at least 100 cells were obtained from duplicate wells in three technical repeats and apoptosis calculated by manual counting of cells with characteristic condensed or pyknotic nuclei. PI was used to exclude necrosis.

Arhoma A.A., Southan J., Chantry A., Haywood‐Small S., & Cross N.A. (2021). EZH2 inhibition enhances TRAIL responses in multiple myeloma but not in quiescent cells.

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Cellular Uptake of FITC-Labeled Peptide

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The cellular uptake of the FITC-labeled peptide was examined in A549 cell line. A549 cells were seeded with RPMI 1640 medium on coverslips in 6-well plates and allowed to adhere overnight. Then the medium was removed and washed with PBS. The cells were treated with FITC-labeled peptide for 1.5 h at 37 °C. After incubation, the media containing the compound were removed and the cells were washed with PBS three times. Coverslips were placed on a microscope slide (Olympus IX81 fluorescence microscope, Olympus Optical Co., Ltd., Tokyo, Japan).

Zakeri-Milani P., Mussa Farkhani S., Shirani A., Mohammadi S., Shahbazi Mojarrad J., Akbari J, & Valizadeh H. (2017). Cellular uptake and anti-tumor activity of gemcitabine conjugated with new amphiphilic cell penetrating peptides. EXCLI Journal, 16, 650-662.

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