Purchased from American Tissue Culture Collection (ATCC), NCI-H28 (RRID:CVCL_1555) cells (a cell line from human malignant pleural mesothelioma) were cultured in Roswell Park Memorial Institute-1640 (RPMI-1640) (Thermo Fisher Scientific, Illkirch-Graffenstaden, France) medium supplemented with 2 mM of L-glutamine, 10% heat-inactivated fetal bovine serum, 100 U/mL penicillin, 100 μg/mL streptomycin (Thermo Fisher Scientific, Illkirch-Graffenstaden, France), as previously described [46 (link)]. The H28 cell line had been authenticated using short tandem repeat profiling within the last three years. Cultured cells were incubated at 37 °C in a humidified atmosphere with 5% CO2. For imaging experiments, H28 cells were plated at a density of 0.4 × 104 cells per cm2 on 35-mm glass bottom microwell dishes (MatTek Corporation, Ashland, MA, USA). All experiments were performed with mycoplasma-free cells.
35 mm glass bottom microwell dishes
Manufactured by MatTek
Sourced in United States
The 35 mm glass bottom microwell dishes are a versatile lab equipment designed for cell culture applications. They feature a transparent glass bottom that allows for high-resolution microscopic imaging of cells. The dishes provide a controlled environment for culturing cells and can be used in a variety of experimental setups.
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Lab products found in correlation
Rpmi 1640, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Cepia er plasmid, by Addgene (2 mentions)
Nucleofector 1 device, by Lonza (2 mentions)
S3e cell sorter, by Bio-Rad (2 mentions)
Nucblue live cell stain, by Thermo Fisher Scientific (2 mentions)
Dulbecco modified eagle medium (dmem), by Lonza (2 mentions)
Rnaimax transfection reagent, by Thermo Fisher Scientific (2 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Plan apochromat 20 0.8 m27 objective, by Zeiss (1 mentions)
Sp5 lscm, by Leica (1 mentions)
Live dead baclight bacterial viability kit, by Thermo Fisher Scientific (1 mentions)
Vpi 1004, by Lonza (1 mentions)
Tcs sp8 sted confocal microscope, by Leica (1 mentions)
Mycoalert detection kit, by Lonza (1 mentions)
Effectene reagent, by Qiagen (1 mentions)
Sigenome non targeting sirna pool 1, by GE Healthcare (1 mentions)
Lsm 710 microscope, by Zeiss (1 mentions)
Eclipse ti2 inverted microscope, by Nikon (1 mentions)
23 protocols using 35 mm glass bottom microwell dishes
1
Culturing and Characterizing NCI-H28 Mesothelioma Cells
2
Visualization of MtDef5 Uptake by F. graminearum
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F. graminearum conidia germlings (50 µL of 105/mL) were incubated with Dylight550-MtDef5 (50 µL of 3 µM) for 1 h or 2 h at room temperature in 10 mm microwell of 35 mm glass bottom microwell dishes (MatTek Corporation, Ashland, MA). Fungal germlings were fixed in 4% paraformaldehyde and washed twice with PBS. DAPI stain solution was prepared by diluting 300 µM DAPI intermediate in PBS (dilution 1:1,000) to make final concentration 300 nM. Nuclear staining was performed by the addition of 100 µL of 300 nM DAPI for 15 min followed by three washes with PBS and culture dish was mounted immediately on the confocal microscope for imaging. DyLight550-MtDef5 was excited at 550 nm and fluorescence was detected at 560–600 nm and DAPI was excited at 405 nm and fluorescence was detected at 430–461 nm.
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F. graminearum conidia germlings (50 µL of 105/mL) were incubated with Dylight550-MtDef5 (50 µL of 3 µM) for 1 h or 2 h at room temperature in 10 mm microwell of 35 mm glass bottom microwell dishes (MatTek Corporation, Ashland, MA). Fungal germlings were fixed in 4% paraformaldehyde and washed twice with PBS. DAPI stain solution was prepared by diluting 300 µM DAPI intermediate in PBS (dilution 1:1,000) to make final concentration 300 nM. Nuclear staining was performed by the addition of 100 µL of 300 nM DAPI for 15 min followed by three washes with PBS and culture dish was mounted immediately on the confocal microscope for imaging. DyLight550-MtDef5 was excited at 550 nm and fluorescence was detected at 560–600 nm and DAPI was excited at 405 nm and fluorescence was detected at 430–461 nm.
3
CEPIA1er Plasmid Transfection in VSCMs
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Cepia ER plasmid was purchased from Addgene (#58215). The CEPIA1er protein was excited at 543 nm, and emitted fluorescence was measured at wavelengths of 580 nm.
VSCMs were transfected in a Nucleofector I device (Lonza) using the Basic Nucleofector Kit for Primary Mammalian Smooth Muscle Cells (VPI-1004, Lonza) and following the manufacturer’s protocol. Six hundred thousand cells were transfected in the presence of 5-μg of plasmid DNA, plated onto 35-mm glass bottom microwell dishes (MatTek Corporation), and grown for 48 hr before experiments were performed.
VSCMs were transfected in a Nucleofector I device (Lonza) using the Basic Nucleofector Kit for Primary Mammalian Smooth Muscle Cells (VPI-1004, Lonza) and following the manufacturer’s protocol. Six hundred thousand cells were transfected in the presence of 5-μg of plasmid DNA, plated onto 35-mm glass bottom microwell dishes (MatTek Corporation), and grown for 48 hr before experiments were performed.
4
Quantifying NAD(P)H Autofluorescence in Cultured Cells
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Epifluorescence microscopy images were collected to confirm autofluorescence measurements. Preparation included culturing cells as a monolayer in 35 mm glass bottom micro well dishes (MatTek Corporation, Ashland, MA) following the previously mentioned growth protocol. Cells were washed with PBS before imaging. In order to capture endogenous fluorescence of NAD(P)H and morphological cell features, an Axio Observer Z1/7 scope with Plan‐Apochromat 20×/0.8 M27 objective was used (ZEISS, Oberkochen, Germany). Bright field images were obtained using the Axiocam 506‐3 V TL lamp. Autofluorescence of NAD(P)H was captured using a 335–383 nm excitation filter and 420–470 nm emission filter corresponding to the NAD(P)H fluorescence spectrum 63 . NAD(P)H fluorescence images (2000 ms) for untreated HeLa cells as well treated cells after 15 min, 30 min, 60 min, and 180 min of treatment were captured. Images were processed for brightness and contrast (Fiji, www.fiji.sc ). Standard workflow for image‐wide cell segmentation and calculation of mean intensity from objects was followed using CellProfiler (www.cellprofiler.org ) to count number of cells and quantify autofluorescence intensities.
5
Biofilm Viability Analysis by LSCM
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Biofilms (n = 6) were grown in 35 mm glass bottom microwell dishes (MatTek Corporation, U.S.A.) under static conditions at 37°C with 5% CO2, washed twice with HBSS and stained with LIVE/DEAD BacLight Bacterial Viability Kit (Life Technologies, U.S.A.) according to manufacturer instructions. Biofilms were examined immediately using a Leica SP5 LSCM with inverted stand using a 63x oil immersion lens, performing sequential scanning using 0.5 µm sections. Three random fields of view were captured and images were analysed using Leica LCS Software.
6
Cellular Uptake Kinetics of MGNS/DOX
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The HeLa cells (ATCC CCL-2) were cultured in Dulbecco’s modified Eagle medium (DMEM) supplement with 10% fetal bovine serum (FPS) and 1% penicillin-streptomycin solution at 37 °C in a 5% CO2 incubator. The cells were grown on the 35 mm glass bottom microwell dishes (MatTek Corp.) in 2 mL DMEM until DMEM reached about 80% confluence. HeLa cells were cultured in a 35 mm petri dish with a piece of coverglass at the bottom of each chamber in the incubation medium (DMEM) for 24 h for cellular uptake studies. Further, MGNS/DOX was added in the incubation medium at a concentration of 100 μg·mL−1 in 5% CO2 at 37 °C for different incubation time (30 min, 60 min, 90 min and 120 min) under different conditions (with magnet, heat or both magnet and heat). Media was then removed and the cells were washed twice with PBS (pH 7.4), before the coverglass was visualized under a fluorescence microscope. Here we used Image J software to analyze the fluorescence intensity of fluorescence microscopic images of the cells. We have used cells image without DOX for baseline correction and relative fluorescence intensity of treated cells measured by subtraction of baseline. For every measurement fluorescent intensity of five different cells were used, while average three different images were used to analyze ±s.d. and P-value by one-way analysis of variance (ANOVA).
7
VSMC Plasmid Transfection Protocol
8
Measurement of ER Calcium Dynamics in VSMCs
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Cepia ER plasmid was purchased from Addgene (#58215). The CEPIA1er protein was excited at 543 nm, and emitted fluorescence was measured at wavelengths of 580 nm.
VSMCs were transfected in a Nucleofector I device (Lonza, Basel, Switzerland) using the Basic Nucleofector Kit for Primary Mammalian Smooth Muscle Cells (VPI-1004, Lonza) and following the manufacturer’s protocol. 50,000 cells were transfected in the presence of 1 μg of plasmid DNA, plated onto 35-mm glass bottom microwell dishes (MatTek Corporation), and grown for 48 h before experiments were performed.
VSMCs were transfected in a Nucleofector I device (Lonza, Basel, Switzerland) using the Basic Nucleofector Kit for Primary Mammalian Smooth Muscle Cells (VPI-1004, Lonza) and following the manufacturer’s protocol. 50,000 cells were transfected in the presence of 1 μg of plasmid DNA, plated onto 35-mm glass bottom microwell dishes (MatTek Corporation), and grown for 48 h before experiments were performed.
9
Mitochondrial Calcium Imaging with Pericam
10
Characterizing Arrestin Recruitment to M2R
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HEK293 (ATCC) cells lacking endogenous βarr1/2 as described elsewhere52 (link),53 (link) were transiently transfected with a 1:4 DNA ratio of pcDNA-teto-M2R and GFP-βarr1-WT or the −3xD variant, respectively. Cells were not authenticated or routinely tested for mycoplasma. Cells were split into 35 mm glass bottom microwell dishes (MatTek) and 24 h thereafter serum starved for 2 h in MEM media containing 20 mM HEPES (pH7.4) and 1 mg/mL BSA. Cells were incubated with Alexa-650-labeled FLAG-M1 antibody and NucBlue Live cell stain (Invitrogen) for 1 h at 37 °C, washed two times in starvation media, and imaged using confocal microscopy. In parallel, transfected cells split into 6-well plates were stimulated for 30 min at 37 °C +/− 1 μM iperoxo. Cells were immediately placed on ice and kept at 4 °C for the remainder of the experiment. Cells were washed twice with cold phosphate-buffered saline and then detached with 0.05% EDTA. Cells were resuspended in assay buffer (Hanks balanced salt solution, 20 mM HEPES pH 7.4, 3 mM CaCl2, 1 mg/mL BSA) and stained with Alexa-650-labeled FLAG-M1 antibody for 30 min at 4 °C. Cells were washed once with assay buffer before analysis by flow cytometry (Bio-Rad S3e Cell Sorter). Data were analyzed using FlowJo software, gating for GFP-positive singlet cells.
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