Glass bottom 96 well plate

Manufactured by MatTek

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The Glass-bottom 96-well plate is a laboratory equipment designed for cell culture and microscopy applications. It features a transparent glass bottom that allows for high-quality imaging and observation of cells under a microscope.

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Glass-bottom plates (24 citations) 96-well plate (7 citations) Glass bottoms (6 citations) Glass bottom wells (2 citations) Poly-L-lysine coated 96-well glass bottom plates (2 citations)

16 protocols using glass bottom 96 well plate

Neutrophil NET Formation in P. aeruginosa

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Isolated neutrophils, 25,000/well, were incubated with P. aeruginosa MOI 10 in a glass-bottom 96-well plate (MatTek Corporation, Ashland, MA, USA) precoated with poly-D-lysine 1 mg/mL (Sigma-Aldrich/Merck, Darmstadt, Germany) for 1 or 4 h at 37 °C and 5% CO₂, and sterile medium was used as a negative control. As we did not observe any significant difference in NET formation in control conditions (in the absence of P. aeruginosa) between 1 and 4 h (see Supplementary Figure S1B), the control values for 4 h were used for the further analysis.
To study the effect of G-CSF on the capacity of neutrophils to form NETs, neutrophils isolated from the blood of healthy volunteers (n = 5) were challenged with P. aeruginosa MOI 10 in the absence or presence of human G-CSF (Filgrastim HEXAL, Holzkirchen, Germany) at a concentration 10 ng/mL for 4 h.

Decker A.S., Pylaeva E., Brenzel A., Spyra I., Droege F., Hussain T., Lang S, & Jablonska J. (2019). Prognostic Role of Blood NETosis in the Progression of Head and Neck Cancer. Cells, 8(9), 946.

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Imaging rG4 structures in cells

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Cells were seeded in a glass bottom 96-well plate (MatTek, USA) and grew overnight. 10 μM RNA oligonucleotides were hybridized with a 10 μM ISCH-app rG4 imaging probe by heating at 95 °C for 5 min and cooling down to room temperature for 2 h. The mixture was then transfected into cells using Lipofectamine 3000 Transfection Reagent (Invitrogen, USA) for over 4 h. Digital images were recorded using an Olympus FV3000 laser scanning confocal microscope with a 63× objective lens, and analyzed with Imaris software (Bitplane Corp., UK).

Lyu K., Chen S.B., Chan C.Y., Tan J.H, & Kwok C.K. (2019). Structural analysis and cellular visualization of APP RNA G-quadruplex †Electronic supplementary information (ESI) available. See DOI: 10.1039/c9sc02768h. Chemical Science, 10(48), 11095-11102.

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Fibroblast Collagen-Matrigel Contraction Assay

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5 × 10⁴ fibroblasts were embedded in 100 µl of collagen I/Matrigel mix (final concentration of collagen I: 4 mg/ml and Matrigel: 2 mg/ml), seeded in a glass‐bottom 96‐well plate (MatTek), and maintained in DMEM supplemented with 10% FBS for 6 days. The gel area was measured using ImageJ software, and the gel contraction was calculated using the formula 100 × (well diameter−gel diameter)/well diameter as previously described (Albrengues et al, 2014 (link)).

Berestjuk I., Lecacheur M., Carminati A., Diazzi S., Rovera C., Prod’homme V., Ohanna M., Popovic A., Mallavialle A., Larbret F., Pisano S., Audebert S., Passeron T., Gaggioli C., Girard C.A., Deckert M, & Tartare‐Deckert S. (2021). Targeting Discoidin Domain Receptors DDR1 and DDR2 overcomes matrix‐mediated tumor cell adaptation and tolerance to BRAF‐targeted therapy in melanoma. EMBO Molecular Medicine, 14(2), e11814.

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Visualizing Neutrophil Extracellular Traps

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Isolated neutrophils 15,000/well were incubated with P. aeruginosa (MOI 10) in glass-bottom 96-well plate (MatTek Corporation, Massachusetts, U.S.) pre-coated with poly-D-lysine 1 mg/ml (Sigma-Aldrich/Merck, Darmstadt, Germany) for 4 h at +37°C, 5% CO₂, sterile medium was used as a negative control. Samples were fixed with paraformaldehyde (Thermo Fisher Scientific, Massachusetts, U.S.) to the final concentration 4%, permeabilized with Triton X-100 (Sigma-Aldrich/Merck, Darmstadt, Germany) 0.2% containing buffer. Since the visualization of NETs using DNA-intercalating dyes alone has the risk of detection of necrotic cells or the generation of artificial results based on dye-blocking peptides associated with NETs, antibody-based techniques are required to visualize NETs. Antibodies for citrullinated histones detect PAD4-dependent NETosis, but not NETs released through other mechanisms (32 (link)). Therefore, anti-histone 1 antibodies (Merck Millipore, Darmstadt, Germany) were used to detect all NETs. Donkey-anti-mouse-AF564 (Invitrogen, Thermo Fisher Scientific, Massachusetts, U.S.) were used as secondary antibodies. Stainings were mounted with ProLong Gold Antifade Mountant with DAPI (Invitrogen, Thermo Fisher Scientific, Massachusetts, U.S.). Percent of NET-producing cells and NETs length were estimated by microscopy.

Pylaeva E., Bordbari S., Spyra I., Decker A.S., Häussler S., Vybornov V., Lang S, & Jablonska J. (2019). Detrimental Effect of Type I IFNs During Acute Lung Infection With Pseudomonas aeruginosa Is Mediated Through the Stimulation of Neutrophil NETosis. Frontiers in Immunology, 10, 2190.

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NLRP6 Phase Separation and Inflammasome Reconstitution

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One-hundred microliters of a BSA solution (50 mg/mL) were added to a glass-bottom 96-well plate (MatTek) and left overnight. This incubation solution was discarded before starting the assay. For the phase separation of different constructs of NLRP6 (MBP-NLRP6, MBP-NLRP6-mCherry, MBP-NLRP6 mutants or domain and IDR truncations) and RNA, MBP-NLRP6 (15 μM) was mixed with 60-bp dsRNA (15 μM) either with or without TEV (0.5 mg/mL) added at time 0 to reach a final buffer concentration of Tris-HCl (20 mM), pH 7.4, NaCl (110 mM), TCEP (0.5 mM), and BSA (1 mg/mL). For the phase-separation assay of the in vitro-reconstituted inflammasome, MBP-NLRP6 (15 μM) was mixed with 60-bp dsRNA (15 μM), MBP-ASC-TAMRA (7.5 μM) and caspase-1-mTurquoise2 (7.5 μM), with TEV (0.5 mg/mL) added at time point 0. The final reaction buffer was Tris-HCl (20 mM) at pH 7.4, NaCl (115 mM), BSA (0.32 mg/mL), and TCEP (0.5 mM). The phase-separation assays for the dependency of salt, temperature, and protein/RNA concentrations were undertaken under similar conditions.

Shen C., Li R., Negro R., Cheng J., Vora S., Fu T.M., Wang A., He K., Andreeva L., Gao P., Tian Z., Flavell R., Zhu S, & Wu H. (2021). Phase Separation Drives RNA Virus-Induced Activation of the NLRP6 Inflammasome. Cell, 184(23), 5759-5774.e20.

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Mapping MBP-NLRP6 Binding to dsRNA and LTA

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Before the phase diagram experiments, we added 50 mg/ml of BSA solution into the glass-bottom 96-well plate (MatTek) for overnight pre-incubation and discarded the BSA before adding the sample. For the phase diagram of MBP-NLRP6 with 60-bp FAM-dsRNA, we created a 6×6 reaction matrix with 6 concentration points of MBP-NLRP6 in X axis (0, 0.47 μM, 0.94 μM, 1.88 μM, 3.75 μM, 7.5 μM) and 6 concentration points of 60-bp FAM-dsRNA in Y axis (0, 0.47 μM, 0.94 μM, 1.88 μM, 3.75 μM, 7.5 μM). The final buffer components were Tris-HCl (20 mM) at pH 7.4, NaCl (156 mM), TCEP (0.6 mM), and BSA (1 mg/mL). The conditions for the phase diagram of MBP-NLRP6 and LTA-TAMRA were similar except that LTA-TAMRA had 6 concentration points of 0, 0.24 μM, 0.47 μM, 0.94 μM, 1.88 μM, 3.75 μM. After adding all the components into the wells, the plate was incubated for 45 minutes at 37 °C. Images were obtained using an ImageXpress Micro Confocal (Molecular Devices) using a 40X objective (S Plan Fluor, NA=0.6)

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Tracking Extracellular Vesicle Uptake Dynamics

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Cells were plated in glass bottom 96-well plates (Mattek; Ashland, MA) analyzing 3 wells per condition. For imaging, recipient cells were labeled with the cytoplasmic dye CellTrackerTM Red (1:1000; ThermoFisher, Waltham, MA) and the nuclear dye Hoechst 33342 (Thermofisher). To enable tracking of EV uptake, EVs were labeled with ExoGlow (Systems Biosciences, EXOC300A-1) according to the manufacturer’s instructions, with excess label being removed by resuspending the labeled EV pellet in PBS, followed by repurification with ExoQuick as published previously [20 ,27 ]. Live-cell imaging of EVs was performed 2 h after adding EVs (5.2 × 10⁸) using the UltraViewVoX spinning disk confocal microscope (Eclipse Ti, Nikon, Tokyo, Japan), running Volocity software (Perkin Elmer, Wokingham, UK) as published previously [18 (link),20 ]. The particle analyzer module in ImageJ was used to detect and quantify the EV (green) - CellTracker (red) colocalized particles (yellow), and the number of EVs is expressed as the ratio of internalized experimental EVs when compared to control EVs.

Nicholson C., Ishii M., Annamalai B., Chandler K., Chwa M., Kenney M.C., Shah N, & Rohrer B. (2020). J or H mtDNA haplogroups in retinal pigment epithelial cells: Effects on cell physiology, cargo in extracellular vesicles, and differential uptake of such vesicles by naïve recipient cells. Biochimica et biophysica acta. General subjects, 1865(4), 129798.

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Live Cell Imaging of C3aR-GFP Transfected Cells

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For imaging, medium was replaced to phenol red free DMEM (Thermo Fisher), with 20 mM HEPES (pH 7.3–7.4). Live cell imaging of C3aR-GFP transfected cell was carried out on glass bottom 96-well-plates (Mattek, Ashland, MA), with cells labeled with plasma membrane dye Wheat Germ Agglutinin (WGA) Deep Red (5 μg/mL; Thermo Fisher), mitochondrial maker MTDR (1 μM; Thermo Fisher) and nuclear marker Hoechst33342 (0.5 μg/mL; Thermo Fisher). Image acquisitions were performed using the UltraViewVoX spinning disk confocal microscope (Eclipse Ti, Nikon, Tokyo JPN), running Volocity software (Perkin Elmer, Wokingham UK) as published (21 (link)). Temperature and cellular pH were maintained using a table top incubator. Images were processed using ImageJ software (NIH). Some chemicals, dynasore, a dynamin inhibitor (200 μM; Sigma-Aldrich,), MnTBAP chloride, a superoxide dismutase mimetic (100 μM; Sigma-Aldrich) and CID1067700, an inhibitor of nucleotide binding by Ras-related GTPases (= Rab7 inhibitor; 100 nM; Sigma-Aldrich) were applied.

Ishii M., Beeson G., Beeson C, & Rohrer B. (2021). Mitochondrial C3a Receptor Activation in Oxidatively Stressed Epithelial Cells Reduces Mitochondrial Respiration and Metabolism. Frontiers in Immunology, 12, 628062.

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Transfection and Imaging of HP1α in NIH 3T3 Cells

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NIH 3T3 cells (ATCC CRL-1658) were seeded in 6-well plates to a density of 2x10 5 cells/well and left to attach overnight at 37°C in 1.5 ml DMEM-Glutamax supplemented with 10% FBS (Gibco) and penicillinstreptomycin (10 µg/ml, Gibco). The following day, medium was replaced with 1.5 ml DMEM-Glutamax supplemented with 10% FBS (Gibco). Lipofectamine 2000 (Invitrogen) was used for transfection following manufacturer's instructions. Transfection reaction used 12 µg of pcDNA3-mEOS3.2-HP1α plasmid and a ratio of 1:4 µg DNA to Lipofectamine 2000. After 4 h incubation, the medium was replaced and left overnight at 37°C. The following day, cells were detached using Trypsin-EDTA and counted before being seeded into glass bottom 96-well plates (Mattek) at density of 5x10 3 cells/well in 100 µl DMEM-Glutamax supplemented with 10% FBS (Gibco) and penicillin-streptomycin (10 µg/ml, Gibco). Cells were left to attach overnight at 37°C. Cells were washed with 100 µl warm PBS (Gibco) and incubated with probe (5 µM) while being co-incubated with endosolytic peptide 4 to help cellular uptake, after cells were washed with incubated with DMSO diluted probe in Optimem medium (Gibco).

Guidotti N., Eördögh Á.M., Mivelaz M., Rivera‐Fuentes P., & Fierz B. (2021). Multivalent peptide ligands to probe the chromocenter microenvironment in living cells.

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Imaging 2H-K2-Pro and 2H-K4NMeS in DM1 Myotubes

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DM1 myotubes were grown in a Mat-Tek 96-well glass bottom plate, differentiated and treated as described above. Cells were then treated with compounds 2H-K2-Pro and 2H-K4NMeS (at 5 μM). After 24 h, the growth medium was removed, the cells were washed twice with 100 μL of 1× DPBS and imaged in 100 μL of Gibco FluoroBrite DMEM. The cells were then imaged using an Olympus Fluoview 1000 confocal microscope at 100× magnification. n = 3 replicates; 1 independent experiment.

Benhamou R.I., Abe M., Choudhary S., Meyer S.M., Angelbello A.J, & Disney M.D. (2020). Optimization of the linker domain in a dimeric compound that degrades an r(CUG) repeat expansion in cells. Journal of medicinal chemistry, 63(14), 7827-7839.

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