Diaphot 300 microscope

Manufactured by Nikon

Sourced in Germany, Japan

The Diaphot 300 is an inverted microscope designed for cell culture and life science applications. It features a sturdy and compact body, a long working distance, and a stable mechanical stage. The Diaphot 300 provides high-quality imaging capabilities for a variety of biological samples.

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Lab products found in correlation

4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions) Ds fi1 camera, by National Instruments (1 mentions) Hoechst 33258, by Thermo Fisher Scientific (1 mentions) Hepes, by Kitazato (1 mentions) Ultra low attachment 6 well plate, by Corning (1 mentions) Facscalibur flow cytometry, by BD (1 mentions) Anti human cd24 pe, by BD (1 mentions) Anti human cd44 fitc, by BD (1 mentions) Interleukin 6 (il 6), by Thermo Fisher Scientific (1 mentions) Epidermal growth factor (egf), by Merck Group (1 mentions) Cellquest software, by BD (1 mentions) Cd24 alexa 647, by BD (1 mentions) Dxm1200, by Nikon (1 mentions) Bacterial sphingomyelinase, by Merck Group (1 mentions) C6 ceramide, by Merck Group (1 mentions) D glucose solution, by Merck Group (1 mentions) Dako fluorescence mounting medium, by Agilent Technologies (1 mentions)

Spelling variants of this product

Diaphot 300 (111 citations) Diaphot (102 citations) Diaphot microscope (61 citations) Diaphot 300 inverted microscope (6 citations) Diaphot 300 fluorescent microscope (4 citations) DIAPHOT- 300 epi-fluorescence microscope (3 citations) Diaphot 300 fluorescence microscope (3 citations) DIAPHOT 300 phase contrast microscope (2 citations)

14 protocols using diaphot 300 microscope

Quantification of Myosin Heavy Chain Expression in Myotubes

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For myosin heavy chain (MHC) staining, cells were fixed with 4% PFA and permeabilized with 0.1% TritonX100 in PBS. After blocking with 5% HS for 1 h, myotubes were incubated overnight with a primary mouse anti-skeletal myosin antibody (MF20 hybridoma) in DPBS. Subsequently, primary antibodies were removed by washing with DPBS. Then, myotubes were incubated with an Alexa 488-conjugated rabbit anti-mouse secondary antibody (Life Technologies, MA, USA), diluted 1:1000, and stained with DAPI (Sigma Aldrich). For imaging, the Nikon Diaphot 300 microscope with 10× magnification was used.
The pictures’ overlay was created using the software Photoshop CS6 (Version CS6, Adobe Inc., CA, USA); brightness and contrast were adjusted to the same degree in every sample group. MHC positive myotubes containing ≥2 nuclei were encircled to quantify the myotube area using the Software Image J 2.0.0 Java 1.6 (NIH). The total number of cell nuclei was counted automatically. For each experiment, six random sections were analyzed.
Micrographs of the MHC-stained cells were also used to determine the fusion index, according to Miersch et al. [48 (link)]. The number of nuclei in the fused multinucleated cells (≥2 nuclei) was divided by the total number of visible nuclei for six randomly chosen pictures per sample and multiplied by 100 to calculate the fusion index.

Petkov S., Brenmoehl J., Langhammer M., Hoeflich A, & Röntgen M. (2022). Myogenic Precursor Cells Show Faster Activation and Enhanced Differentiation in a Male Mouse Model Selected for Advanced Endurance Exercise Performance. Cells, 11(6), 1001.

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Wound Healing Assay in MDA-MB-231 Cells

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MDA-MB-231 cells were allowed to grow confluently in 12-well plates. A linear wound was created by scraping the wells with a micropipette tip. The floating cells were removed by gentle washes in culture medium, and then the cells were cultured with completed media in the presence of 7 at 100 µM or 1% DMSO. The degree of wound closure was assessed using a Nikon Diaphot 300 microscope in three randomly chosen regions by measuring the distance between the wound edges just after wounding and after 16 h.

Liu D., Zhou D., Wang B., Knabe W.E, & Meroueh S.O. (2015). A New Class of Orthosteric uPAR•uPA Small-Molecule Antagonists Are Allosteric Inhibitors of the uPAR•Vitronectin Interaction. ACS chemical biology, 10(6), 1521-1534.

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Evaluating Anti-angiogenic Potential of Compounds

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The HMVEC/NHDF co-culture was seeded in 96-well plates in co-culture medium and incubated for 24 hours. The medium was then removed and replaced by co-culture medium containing, or not (control), dextran sulfate (10, 30, and 100 µg/mL) or the positive reference (suramin 100 µM) and then the cells were stimulated with VEGF (100 ng/mL). In parallel, a non-stimulated control was performed. Cells were incubated for 7 days with treatment renewal after 72 hours of incubation. After incubation, the co-culture medium was discarded and the cells were rinsed, fixed, permeabilized, and labeled using an anti-collagen IV primary antibody. The primary antibody was then revealed using an appropriate fluorescent secondary antibody (GAR-Alexa 568), and the cell nuclei were stained in parallel using Hoechst 33,258 solution (bis-benzimide). The formation of pseudotubes was observed using a NIKON Diaphot 300 microscope (objective lens ×4). Images were captured using a NIKON DS-Fi1 camera and NIS-Elements 4.13.04 software. The analysis of pseudotube formation was performed through collagen IV labeling using Image J software. The percentage inhibition of VEGF-induced pseudotube formation was calculated using the mean of the pseudotube area (mm²) in the different conditions.

Hernandez-Pigeon H., Garidou L., Galliano M.F., Delga H., Aries M.F., Duplan H., Bessou-Touya S, & Castex-Rizzi N. (2018). Effects of dextran sulfate, 4-t-butylcyclohexanol, pongamia oil and hesperidin methyl chalcone on inflammatory and vascular responses implicated in rosacea. Clinical, Cosmetic and Investigational Dermatology, 11, 421-429.

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Paclitaxel Solubility Dynamics Imaged by DIC

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For DIC microscopy, a total of 1 μL of a 500, 400, 300, 200, 150, or 100 μM solution of PTX in DMSO was added to 9 μL of an unfiltered suspension of CM pellet diluted to ≈10¹¹ particles/mL (per NTA) in deionized water, or deionized water alone in small PCR tubes. These samples were incubated for 24 h at room temperature before imaging. After incubation, 1 μL aliquots were placed on glass microscope slides and covered by a coverslip kept in place by parafilm cutouts. These slides were imaged at 20× magnification on an inverted Diaphot 300 microscope (Nikon). The kinetic phase diagrams of PTX solubility are based on the results of two independently prepared samples.

Fisher W.S., Tchounwou C., Wei S., Roberts L., Ewert K.K, & Safinya C.R. (2021). Exosomes are secreted at similar densities by M21 and PC3 human cancer cells and show paclitaxel solubility. Biochimica et biophysica acta. Biomembranes, 1864(4), 183841.

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Apoptosis Quantification in Osteoclasts

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Osteoclasts on glass coverslips were fixed with 4% paraformaldehyde and stained for TUNEL (Roche Diagnostics, Indianapolis, IN) according to the manufacturer's protocols for 30 min at 37°C. After incubation, coverslips were washed and stained with Hoechst 33258 (Invitrogen) for 30 min. Coverslips were visualized on a Nikon Diaphot 300 microscope. TUNEL-positive, condensed nuclei were identified as apoptotic. TUNEL analysis for apoptosis was accomplished by counting a total of 100 osteoclasts per coverslip. Data shown is from an average of 4 independent experiments with H₂O₂ treatment and 3 independent experiments with rotenone.

Sharma R., Callaway D., Vanegas D., Bendele M., Lopez-Cruzan M., Horn D., Guda T., Fajardo R., Abboud-Werner S, & Herman B. (2014). Caspase-2 Maintains Bone Homeostasis by Inducing Apoptosis of Oxidatively-Damaged Osteoclasts. PLoS ONE, 9(4), e93696.

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BV-2 Cell Culture and Botanical Compound Treatment

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The murine BV-2 cell line was generated by infecting primary microglia cell cultures with a v-raf/v-myc oncogene carrying retrovirus (J2) [28 (link)]. These cells were obtained as a gift from Dr. R. Donato [29 (link)] and prepared as previously described [30 (link)]. Cells were cultured in 75 cm² flasks with DMEM supplemented with 5% FBS containing 100 units/ml penicillin and100 μg/ml streptomycin, and maintained in 5% CO₂ incubator at 37°C. Normally, BV-2 cells were grown in 100 mm dish and after 80–90% confluent, they were removed and subcultured in 6-, 12-, or 96-well plates, depending on the experiment. Cells were then cultured overnight or until 80–90% confluent. Cells were serum starved for 3 h followed by adding botanical compounds (quercetin 0–20 μM or cyanidin, 0–100 μM) or enzyme inhibitors (0–10 μM) for 1 h and then stimulated with LPS (0–200 ng/ml). Test compounds were dissolved in DMSO and added to culture at concentrations of less than 0.5%. In most experiments, cell culture was tested to ensure the levels of DMSO exerted no deleterious effects on the responses. In some studies, cell morphology was examined using a phase contrast Nikon DIAPHOT 300 microscope attached with a CCD cool camera linked to the MagnaFire 2.1C software for image processing.

Sun G.Y., Chen Z., Jasmer K.J., Chuang D.Y., Gu Z., Hannink M, & Simonyi A. (2015). Quercetin Attenuates Inflammatory Responses in BV-2 Microglial Cells: Role of MAPKs on the Nrf2 Pathway and Induction of Heme Oxygenase-1. PLoS ONE, 10(10), e0141509.

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Evaluating Testicular Function in Mice

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Testis and epididymides were carefully dissected. Bilateral testes were weighed and frozen in liquid nitrogen, and then processed for real-time PCR, western blot analysis or caspase-6 activity assay. Separated cauda epididymis from each mouse was immediately placed into modified HTF medium with HEPES (KITAZATO, #93421) at room temperature. The sperm number in the suspension was counted using a hemocytometer on a Nikon DIAPHOT 300 microscope at 200× magnification. At least 200 sperm were counted in each sperm sample. We assessed motility rate as the percentage of the sum of sperms with progressive motility and non-progressive motility per total sperm number.

Kobori T., Iwabu M., Okada-Iwabu M., Ohuchi N., Kikuchi A., Yamauchi N., Kadowaki T., Yamauchi T, & Kasuga M. (2024). Decreased AdipoR1 signaling and its implications for obesity-induced male infertility. Scientific Reports, 14, 5701.

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Senescence Assay in MEFs

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Hat1^+/+, Hat1^+/−, and Hat1^−/− MEFs were seeded in 2‐well chamber slides (Lab‐Tek). MEFs were stained with β‐galactosidase staining kit according to the manufacturer's protocol (Invitrogen). Stained slides were visualized with a Nikon DIAPHOT 300 microscope, and images were captured using imaging software for microscopy SPOT 5.3. The percentage of senescent cells was calculated as the total number of β‐galactosidase staining cells divided by the total number of cells.

Nagarajan P., Agudelo Garcia P.A., Iyer C.C., Popova L.V., Arnold W.D, & Parthun M.R. (2019). Early‐onset aging and mitochondrial defects associated with loss of histone acetyltransferase 1 (Hat1). Aging Cell, 18(5), e12992.

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Mammosphere Formation and Characterization

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A single-cell suspension was cultured on 6-well ultralow-attachment plate (Corning) at a density of 4 × 10⁴ cells/well in serum-free DMEM/F12 medium with 1% L-glutamine, 1% penicillin/streptomycin, 2% B27 (Invitrogen), 20 ng/ml EGF (Sigma-Aldrich) and 20 ng/ml FGFb (PeproTech). IL-6 (50 μg/ml) (PeproTech) or tocilizumab (200 μg/ml) was added to examine the mammosphere formation. Mammospheroids were photographed at a magnification of 200× using a Nikon DIAPHOT300 microscope at the indicated time. Mammosphere cells (1 × 10⁵) were further stained with anti-human CD24-PE (BD Pharmingen), anti-human CD44-FITC (BD Pharmingen), CD24-Alexa 647 (BD Pharmingen) or EpCAM-BB515 (BD Pharmingen) for 1 h at 4 °C, PBS rinsed and resuspended in 500 μl PBS. CD44-FITC and EpCAM-BB515 were excited at 490 nm, and the emissions were determined by FL1 PMT (515–545 nm bandpass filter). CD24-Alexa 647 was excited at 633 nm, and the emissions were determined by FL-4 PMT (653–669 nm bandpass filter). BD FACSCalibur flow cytometry (BD Bioscience) and Cell Quest software (BD Biosciences) were used to identify CD44(+)/CD24(−) and EpCAM(+) subpopulations.

Weng Y.S., Tseng H.Y., Chen Y.A., Shen P.C., Al Haq A.T., Chen L.M., Tung Y.C, & Hsu H.L. (2019). MCT-1/miR-34a/IL-6/IL-6R signaling axis promotes EMT progression, cancer stemness and M2 macrophage polarization in triple-negative breast cancer. Molecular Cancer, 18, 42.

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Quantifying Neuronal Calcium Dynamics and Gene Expression

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All results are expressed as mean ± SEM. An unpaired t-test was used for RT-PCR of DRG primary culture neurons and the calcium imaging data to compare the groups. A one-way ANOVA was used for the RT-PCR of CFA plantar injected skin and DRG primary culture with the p-p38 inhibitor. For all behavioral testing, a two-way ANOVA followed by Bonferroni posttests was used. Differences were accepted as significant if the probability was less than 5% (p < 0.05). All emulsion-coated slides were digitized with a Nikon DIAPHOT-300 microscope connected to a Nikon DXM1200 digital camera. We used Adobe Photoshop Element 10.0 (Adobe Systems, Mountain View, CA, USA) to optimize the images and prepare all figures.

Ikeda-Miyagawa Y., Kobayashi K., Yamanaka H., Okubo M., Wang S., Dai Y., Yagi H., Hirose M, & Noguchi K. (2015). Peripherally increased artemin is a key regulator of TRPA1/V1 expression in primary afferent neurons. Molecular Pain, 11, 8.

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