Plate 96 well black plate

Manufactured by Ibidi

Sourced in Germany

The µ-Plate 96-well black plates are a type of microplate designed for various cell-based assays. Each plate contains 96 individual wells with a black background color.

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Lab products found in correlation

Lsm 700 confocal microscope, by Zeiss (2 mentions) Sulfinpyrazone, by Merck Group (1 mentions) Mitotracker red cm h2xros, by Thermo Fisher Scientific (1 mentions) Fluo 4 acetoxymethyl ester, by Thermo Fisher Scientific (1 mentions) Pluronic f 127, by Merck Group (1 mentions) Mitophagy detection kit, by Dojindo Laboratories (1 mentions) Live imaging solution, by Thermo Fisher Scientific (1 mentions) Fluoro carbonyl cyanide phenylhydrazone (fccp), by Merck Group (1 mentions) Fluosphere carboxylate modified microspheres, by Thermo Fisher Scientific (1 mentions) Zen 2011 sp3, by Zeiss (1 mentions) Wga cf488a, by Biotium (1 mentions) 1 1 dioctadecyl 3 3 3 3 tetramethylindocarbocyanine perchlorate dii, by Thermo Fisher Scientific (1 mentions) Ca2 and mg2, by Merck Group (1 mentions)

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96-well plates

4 protocols using plate 96 well black plate

Mitochondrial and Calcium Imaging in hiPSC-CMs

Cited 1 time

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hiPSC-CMs were differentiated on µ-Plate 96-well black plates (Ibidi), experiments were carried out in an extracellular medium, images were acquired and quantified as previously described [1 (link), 61 (link)].
To monitor mitochondrial ROS and mitochondrial membrane potential, cells were incubated with 25 nM MitoTracker Red CM-H₂XRos, λ_exc 580 nm and λ_em 600 nm (MTR, Thermo Fisher Scientific), or 25 nM tetramethylrhodamine, λ_exc 550 nm and λ_em 580 nm (TMRM, Thermo Fisher Scientific), respectively. Images were collected at four different time points.
To monitor cytosolic Ca²⁺ transients, cells were loaded with Fluo-4 acetoxymethyl ester, λ_exc 490 nm and λ_em 515 nm (Thermo Fisher Scientific), 0.01% w/v pluronic F-127 (Sigma), and 250 µM sulfinpyrazone (Sigma). To evaluate the release of Ca²⁺ from the sarcoplasmic reticulum (SR), a pulse of 10 mM caffeine was added to the cells.
To monitor mitochondrial Ca²⁺, hiPSC-CMs were infected with an adenovirus containing mito-GCaMP5G [38 (link)] and 48 h later imaged both at baseline and following a pulse of 10 mM caffeine. Results are expressed as fluorescence intensity ratio F_λ1/F_λ2, in which λ₁ (~ 410 nm) and λ₂ (~ 480 nm), respectively.

Di Sante M., Antonucci S., Pontarollo L., Cappellaro I., Segat F., Deshwal S., Greotti E., Grilo L.F., Menabò R., Di Lisa F, & Kaludercic N. (2023). Monoamine oxidase A-dependent ROS formation modulates human cardiomyocyte differentiation through AKT and WNT activation. Basic Research in Cardiology, 118(1), 4.

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Monitoring Mitophagy in hiPSC-Derived Cardiomyocytes

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To monitor mitophagy levels in hiPSC-CMs, cells were plated on µ-Plate 96-well black plates (Ibidi) and the mitophagy detection Kit (Dojindo) was used. Briefly, cells were washed twice with PBS and incubated with 100 nmol/l Mtphagy Dye working solution at 37 °C for 30 min. Subsequently, the supernatant was discarded and wells washed twice with PBS. To induce mitophagy, positive control groups were incubated with FCCP (Sigma-Aldrich) 10 µM at 37 °C for 120 min. At the end of the induction time, medium was removed and cells washed twice with PBS. To observe the co-localization of Mtphagy Dye and lysosome, cardiomyocytes were incubated at 37 °C for 30 min with 1 μmol/l Lyso Dye working solution. Images were collected in live imaging solution (Thermo) using Crest X-light V3 confocal system and quantification of co-localization was determined using ImageJ FIJI. Three independent experiments were performed and at least ten fields of view per experiment were quantified.

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Fluorescent Labeling of Pollen Aggregates

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The pollen aggregates collected in PBS was soaked in PBS containing FluoSphere Carboxylate-Modified Microspheres, 0.5 µm, red fluorescent (580/605) (#F8812, ThermoFisher) and/or WGA-CF488A (Biotium) in a well of µ-Plate 96 Well Black plate (#89626, ibidi, Germany). The fluorescence was observed under the LSM 700 confocal microscope (Zeiss) using ZEN2011 SP3 software (Zeiss).

Matsuzawa M., Ando T., Fukase S., Kimura M., Kume Y., Ide T., Izawa K., Kaitani A., Hara M., Nakamura E., Kamei A., Matsuda A., Nakano N., Maeda K., Tada N., Ogawa H., Okumura K., Murakami A., Ebihara N, & Kitaura J. (2023). The protective role of conjunctival goblet cell mucin sialylation. Nature Communications, 14, 1417.

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Membrane Staining of CHO-K1-hM4R Cells

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CHO-K1-hM₄R cells were grown as described above and seeded with a density of 25 000 cells per well into a µ-Plate 96 well Black plate (Ibidi, Gräfelfing, Germany) 5 h before the experiment. A stock solution of 1 mM 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI) (Invitrogen, Eugene, Oregon, USA) in DMSO stored at −20°C was thawed and sonicated in an ultrasound bath for 5 min to disrupt aggregates. Cell medium was removed and replaced with 200 µl per well of 2 µM DiI in Dulbecco's phosphate-buffered saline (DPBS) with Mg²⁺ and Ca²⁺ (Sigma-Aldrich) to stain the cell membranes. The cells were incubated with the solution for 10 min before imaging. The cells were imaged with Cytation 5 cell imaging multi-mode plate reader equipped with 20X LUCPLFLN objective (Olympus) from Bright-field and RFP channels (LED light source with excitation filter 531(40) nm and emission filter 593(40) nm for RFP channel (BioTek Instruments) with the following parameters for bright-field: LED intensity = 4, integration time = 110 ms, camera gain = 24 and for RFP fluorescence channel: LED intensity = 1, integration time = 71 ms, camera gain = 24. The cells were imaged in the montage mode (196 locations) with Z-stack (10 planes, 4 planes below and 5 planes above focus) to cover any imaging location-dependent variability and simulate potential autofocusing errors.

Tahk M.J., Torp J., Ali M.A., Fishman D., Parts L., Grätz L., Müller C., Keller M., Veiksina S., Laasfeld T, & Rinken A. (2022). Live-cell microscopy or fluorescence anisotropy with budded baculoviruses—which way to go with measuring ligand binding to M4 muscarinic receptors?. Open Biology, 12(6), 220019.

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