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D35c4 20 1.5 n

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D35C4-20-1.5-N is a laboratory equipment product manufactured by Cellvis. It is a 35 mm culture dish with a 20 mm central well and a 1.5 mm depth. The 'N' in the product code indicates that it is a non-treated dish.

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Lab products found in correlation

Poly d lysine, by Thermo Fisher Scientific (4 mentions) Live cell imaging solution, by Thermo Fisher Scientific (4 mentions) Sytox green, by Thermo Fisher Scientific (3 mentions) Mitopy1, by Merck Group (2 mentions) Lipofectamine 3000 reagent, by Thermo Fisher Scientific (2 mentions) Phorbol 12 myristate 12 acetate pma, by Merck Group (2 mentions) Dulbecco modified eagle medium (dmem), by Merck Group (2 mentions) Thp 1, by American Type Culture Collection (2 mentions) Raw264, by American Type Culture Collection (2 mentions) Amaxa nucleofector kit, by Lonza (2 mentions) Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions) Bodipy 581 591 c11, by Thermo Fisher Scientific (1 mentions) Plan apochromat 20x 0.8 m27 objective, by Zeiss (1 mentions) Lsm 880 microscope, by Zeiss (1 mentions) Cal 590 am, by AAT Bioquest (1 mentions) Fm4 64, by Thermo Fisher Scientific (1 mentions) Mitotracker green fm, by Thermo Fisher Scientific (1 mentions) Lsm 880 airyscan confocal microscope, by Zeiss (1 mentions) Image it tmrm reagent, by Thermo Fisher Scientific (1 mentions) W1 spinning disk microscope, by Nikon (1 mentions)

17 protocols using d35c4 20 1.5 n

1

Evaluating Lipid Peroxidation via Flow Cytometry and Confocal Microscopy

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Lipid peroxidation was analyzed by flow cytometry and confocal laser-scanning microscope. For flow cytometry analysis, cells were plated in six-well culture plate overnight and then treated with indicated agents. Next, cells were incubated with 10 μM BODIPY 581/591 C11 (Thermo Fisher, D3861) for 30 min, washed with PBS, trypsinized and resuspended in PBS, and then subjected to flow cytometry analysis. For confocal imaging, cells were seeded in a 4-chamber glass bottom dish (Cellvis, D35C4-20-1.5-N) and treated as previously indicated. After incubated with 5 μM BODIPY 581/591 C11 for 30 min and washed with PBS, images were acquired using the confocal laser-scanning microscope.
Tang S., Shen Y., Wei X., Shen Z., Lu W, & Xu J. (2022). Olaparib synergizes with arsenic trioxide by promoting apoptosis and ferroptosis in platinum-resistant ovarian cancer. Cell Death & Disease, 13(9), 826.
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2

Calcium Imaging of Pancreatic Islets

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Islets from GKKI and control mice were left to recover overnight in Islet Medium (RPMI 1640, 11mM Glucose, 10% FBS, with Penicillin-Streptomycin 100 IU-μg/ml). On the day of the experiment, 8 islets per group were transferred to a Petri dish with Krebs-Ringer Bicarbonate buffered medium + 10 mM HEPES (KRBH) at pH 7.40, supplemented with + 0.1 % Bovine Serum Albumin and 1 mM glucose. The cell permeant calcium indicator Cal-590 AM (AAT Bioquest, Inc.) was added to the dish at a concentration of 4 μM, and the dish was set on a shaker at room temperature for 30 minutes. Next, the islets were transferred to a 4-chamber glass bottom dish (D35C4–20-1.5-N, Cellvis) containing KRBH with 0.1 % BSA and 1 mM glucose, and the dish was placed in a stage-top incubator set at 37°C, 5% CO2, 100% humidity for the duration of the experiment. Imaging was performed using a Zeiss LSM880 microscope with a Plan-Apochromat 20x/0.8 M27objective, with the following settings: 561 nm laser excitation, 570–695 nm bandpass filter, pixel size 1.661 μm, pixel dwell time 33.0 μs. After each glucose concentration was added, we waited 6 minutes before starting the 5-minute time-course with one image every 5 seconds. For the study of calcium waves across islets, we employed a pixel size of 1.371 μm and a pixel dwell time of 3.45 μs to acquire images every 42 ms.
Chen K.H., Doliba N., May C.L., Roman J., Ustione A., Tembo T., Negron A., Radovick S., Piston D.W., Glaser B., Kaestner K.H, & Matschinsky F.M. (2022). Genetic Activation of Glucokinase in a Minority of Pancreatic Beta Cells Causes Hypoglycemia in Mice. Life sciences, 309, 120952.
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3

Imaging of Mitochondrial Function in HEK293T Cells

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250,000–300,000 HEK293T cells/well were plated in 700 µL DMEM glutamax (10% FBS) into 2 wells of a 4 well chambered imaging dish (D35C4-20-1.5-N, Cellvis), which were precoated with 4 µg Poly-D-lysine (30–70 KDa, Alfa Aesar) for 2 h. After 20–24 h, the media was replaced by 500 µL DMEM glutamax. After 6 h of starvation, the cells were treated for another 6 h with 500 µL of 1% BSA ± 1 mM Palmitate made with DMEM glutamax. Then, the media was replaced by 1 µM Hoechst 33342 and 100 nM MitoTracker Deep Red in 400 µL DMEM glutamax containing 1% BSA ± 1 mM Palmitate. After 30 min of incubation at 37 °C, the cells were washed with 400 µL of Live Cell Imaging Solution and replaced by 1 µM DPP-2/500 nM mitoDPP-2 in Live Cell Imaging Solution (Molecular Probes). After 10 min of incubation at 37 °C, images were obtained as described above with the following settings: for DPP-2 (exposure time 150 or 250 ms, EM gain 100 or 150), mitoDPP-2 (exposure time 150 ms, EM gain 75), MitoTracker Deep Red (exposure time 15 ms, EM gain 15), Hoechst 33342 (exposure time 40 ms, EM gain 20), and brightfield (exposure time 100 ms, EM gain 50). Analyses were performed as described above and each experiment was repeated in at least three biological replicates with identical results.
Kathayat R.S., Cao Y., Elvira P.D., Sandoz P.A., Zaballa M.E., Springer M.Z., Drake L.E., Macleod K.F., van der Goot F.G, & Dickinson B.C. (2018). Active and dynamic mitochondrial S-depalmitoylation revealed by targeted fluorescent probes. Nature Communications, 9, 334.
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4

Quantifying Mitochondrial H2O2 in Cells

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300,000 HEK293T cells/well or 125,000 HepG2 cells/well were plated in four-well chamber slides (D35C4-20-1.5-N, Cellvis) precoated with 5 μg Poly-D-lysine (30-70 KDa, Alfa Aesar). After 20-24 hr, cells were pretreated with 10 μM PalmB, 5 μM ML348, or 2.5 μM mitoFP for 30 min at 37 °C. Control cells were pretreated with vehicle (DMSO). Hoechst 33342 and MitoTracker Deep Red were included for respective nuclear and mitochondrial visualization, as was 2 μM of the mitochondrial-targeted H2O2 probe, mitoPY1 (Sigma). After pretreatment, cells were briefly washed with DPBS, and treated with 100 μM H2O2 in fresh DPBS (400 μL) for 10 min at 37 °C. Control cells were untreated. Cells were then imaged on an inverted epifluorescence microscope. Analyses were performed in ImageJ (Wayne Rasband, NIH). For data analysis, the average fluorescence intensity per image in each experimental condition was obtained by gating cells using the brightfield image and applying that mask in the corresponding mitoPY1 image. All data was normalized to the average fluorescence intensity of the DMSO-pretreated control that was not exposed to H2O2. Each experiment was repeated in at least two biological replicates with identical results.
Cao Y., Qiu T., Kathayat R.S., Azizi S.A., Thorne A.K., Ahn D., Fukata Y., Fukata M., Rice P.A, & Dickinson B.C. (2019). ABHD10 is an S-depalmitoylase affecting redox homeostasis through peroxiredoxin-5. Nature chemical biology, 15(12), 1232-1240.
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5

TSPAN4 Localization in NRK and MGC803 Cells

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NRK cells with stable TSPAN4-GFP expression (NRK-TSPAN4-GFP), WT NRK, WT MGC803 or MGC803-TSPAN4-KO cells were seeded (around 4 × 103 cells) into 3.5 cm glass-bottom confocal dish (Cellvis D35C4-20-1.5-N), which was pre-coated with 10 μg/mL fibronectin (Gibco, Thermo Fisher scientific PHE0023) and grew for 12–15 h. The cells were stained with 5 μg/mL FM4-64 (Invitrogen, Thermo Fisher scientific T3166) for 15 min at 37 °C.
Dharan R., Huang Y., Cheppali S.K., Goren S., Shendrik P., Wang W., Qiao J., Kozlov M.M., Yu L, & Sorkin R. (2023). Tetraspanin 4 stabilizes membrane swellings and facilitates their maturation into migrasomes. Nature Communications, 14, 1037.
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6

Live-cell imaging of mitochondria and membrane potential

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For live cell imaging, cells were grown in four-chamber glass-bottom dishes (D35C4-20-1.5-N; Cellvis). Cells were loaded with MitoTracker Green FM (200 nM; Thermo Fisher) together with Image-iT TMRM reagent (1:1,000; Thermo Fisher) in Live Cell Imaging Solution (Thermo Fisher, catalog no. A14291DJ) at 37°C for 30 min. Cells then were washed three times and imaged in Live Cell Imaging Solution at 37°C and 5% CO2. The cells were analyzed using Zeiss LSM 880 Airyscan confocal microscope (objective Plan-Apochromat 63×/1.4 oil; Zeiss) equipped with a Pecon stage-top incubator with controlled temperature and CO2. Images in each experiment were acquired at identical settings (Ex/Em 561/606, detection wavelength of 571 to 641, detector gain 646.2 for TMRM; Ex/Em 488/530, detection wavelength of 499 to 561, detector gain 837.0 for MitoTracker Green FM).
Ma H., Qian W., Bambouskova M., Collins P.L., Porter S.I., Byrum A.K., Zhang R., Artyomov M., Oltz E.M., Mosammaparast N., Miner J.J, & Diamond M.S. (2020). Barrier-to-Autointegration Factor 1 Protects against a Basal cGAS-STING Response. mBio, 11(2), e00136-20.
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7

Live-cell Confocal Imaging of Dopaminergic Neurons

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Confocal live-cell imaging was performed using a Nikon W1 Spinning Disk microscope with a 100×-oil objective (TIRF 100x 1.49 NA; Nikon Plan Apo). Dopaminergic neurons (day 70) were cultured and transduced or stained as described above and imaged in four-chamber glass-bottom dishes (D35C4-20-1.5-N; Cellvis) in a temperature-controlled (37°C) and a humidified chamber with 5% CO2. Images were acquired in single-camera mode with 500-ms exposure time. Cells were imaged at 1 frame every 2 s for 3 min total. For 3D live-cell imaging, cells were imaged as stated above with z-steps of 0.2 µm. All live-cell confocal images for analysis of Rab10 and VPS13C localization in live COS7 cells were acquired on a Nikon A1R laser scanning confocal microscope with GaAsp detectors using a Plan Apo λ 100x 1.45 NA oil immersion objective (Nikon) using NIS-Elements (Nikon). Live cells were imaged in a temperature-controlled chamber (37 °C) at 5% CO2 at one frame every 2–3 s. Dual-color videos were acquired as consecutive green-red images and tricolor videos were acquired as consecutive green-red-blue images.
Schrӧder L.F., Peng W., Gao G., Wong Y.C., Schwake M, & Krainc D. (2024). VPS13C regulates phospho-Rab10-mediated lysosomal function in human dopaminergic neurons. The Journal of Cell Biology, 223(5), e202304042.
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8

Quantifying Mitochondrial H2O2 in Cells

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300,000 HEK293T cells/well or 125,000 HepG2 cells/well were plated in four-well chamber slides (D35C4-20-1.5-N, Cellvis) precoated with 5 μg Poly-D-lysine (30-70 KDa, Alfa Aesar). After 20-24 hr, cells were pretreated with 10 μM PalmB, 5 μM ML348, or 2.5 μM mitoFP for 30 min at 37 °C. Control cells were pretreated with vehicle (DMSO). Hoechst 33342 and MitoTracker Deep Red were included for respective nuclear and mitochondrial visualization, as was 2 μM of the mitochondrial-targeted H2O2 probe, mitoPY1 (Sigma). After pretreatment, cells were briefly washed with DPBS, and treated with 100 μM H2O2 in fresh DPBS (400 μL) for 10 min at 37 °C. Control cells were untreated. Cells were then imaged on an inverted epifluorescence microscope. Analyses were performed in ImageJ (Wayne Rasband, NIH). For data analysis, the average fluorescence intensity per image in each experimental condition was obtained by gating cells using the brightfield image and applying that mask in the corresponding mitoPY1 image. All data was normalized to the average fluorescence intensity of the DMSO-pretreated control that was not exposed to H2O2. Each experiment was repeated in at least two biological replicates with identical results.
Cao Y., Qiu T., Kathayat R.S., Azizi S.A., Thorne A.K., Ahn D., Fukata Y., Fukata M., Rice P.A, & Dickinson B.C. (2019). ABHD10 is an S-depalmitoylase affecting redox homeostasis through peroxiredoxin-5. Nature chemical biology, 15(12), 1232-1240.
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9

Live-cell Microscopy of Transfected Cell Lines

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HeLa, HEK293T, THP-1, and RAW264.7 cells were obtained from American Type Culture Collection and cultured in Dulbecco’s modified Eagle’s media (DMEM, Sigma-Aldrich, St Louis, MO, USA) or RPMI-1640 medium, supplemented with 10% fetal bovine serum (FBS) under 37 °C at a 5% CO2 atmosphere. Phorbol 12-myristate 12-acetate (PMA) from Sigma Aldrich (St Louis, MO, USA) was used at 20 ng/mL to induce THP-1 differentiation to macrophage. DNA transfections were carried out by using the Lipofectamine 3000 reagent (Life Technologies, Carlsbad, CA, USA) or using an Amaxa nucleofector kit (Lonza) for transfection of plasmids into THP-1 and RAW264.7 cells. For live-cell imaging, cells were seeded in four-chamber 35-mm glass-bottom dishes (D35C4-20-1.5-N, Cellvis, Mountain View, CA, USA) at 40-60% confluency, and imaged 24 hours after transfection.
Tan P., He L, & Zhou Y. (2020). Engineering supramolecular organizing centers for optogenetic control of innate immune responses. Advanced biology, 5(5), e2000147.
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10

Live-Cell Imaging of Transfected Cells

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DNA transfection was performed by using the Lipofectamine 3000 transfection reagent (Thermo Fisher Scientific, MA, USA) according to the manufacturer’s instructions. For live-cell fluorescence imaging experiments, cells were seeded in four-chamber 35-mm glass-bottom dishes (D35C4–20-1.5-N, Cellvis, Mountain View, CA, USA) one day before transfection, and imaged 24–48 h after transfection in an imaging cage equipped with 5% CO2 with the temperature set at 37 °C.
He L., Tan P., Huang Y, & Zhou Y. (2021). Design of Smart Antibody Mimetics with Photosensitive Switches. Advanced biology, 5(5), e2000541.
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