For the adherent SUM1315 cell line, cells were seeded in IbiTreat 8-well μ-slides (Ibidi®, Gräfelfing, Germany) at a concentration of 50 × 103 cells per well. Slides were maintained for 3 days at 37 °C in a humidified incubator in order to obtain homogeneous confluent culture. The suspension DU4475 cells were directly harvested from the culture flask. For both cell lines, cells were fixed with a 4% para-formaldehyde (PFA, Sigma, Darmstadt, Germany) solution before being incubated in a 1 μM fluorescent conjugate PBS solution for 1 h. Three successive washes of 20 minutes were then conducted in PBS before imaging with a Cytation™3 MV (BioTek®, Winooski, VT, USA) fluorescent microscopy module (M = 40×, fluorescence filters = GFP, RFP or Cy5). Cells mean fluorescence intensity was calculated with Gen5 3.08 software (BioTek®) for three independent experiments. For each condition, fluorescence intensity was measured from 10 different imaging fields. For P-gp silencing, SUM1315 cells were exposed 72 hours to specific ABCB1 siRNAs diluted at 50 nM in culture medium and lipofectamine solution (Ambion®, reference AM51331 assay ID #4123/ABCB1, sequences 5′ > 3′: GGAUAUUAGGACCAUAAAUtt (sense), AUUUAUGGUCCUAAUAUCCtg (Antisense)). Cells were then fixed with para-formaldehyde 4% solution and stained with fluorescent conjugates or specific antibodies as previously described.
Ibitreat 8 well μ slides
Manufactured by Ibidi
Sourced in Germany
The IbiTreat 8-well μ-slides are a microscopy-optimized cell culture tool. These slides feature 8 individually coated wells for cell culture and analysis. The wells are designed to provide a high-quality environment for cell growth and imaging.
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Lab products found in correlation
Sp8 confocal microscope, by Leica (3 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions)
Gen5 3, by Agilent Technologies (1 mentions)
Cytation 3 mv, by Agilent Technologies (1 mentions)
Rhodamine phalloidin, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Las x software platform, by Leica (1 mentions)
Fibronectin, by Corning (1 mentions)
Lipofectamine 3000 transfection reagent, by Thermo Fisher Scientific (1 mentions)
Ab153915, by Abcam (1 mentions)
Mitotracker orange cmtmros, by Thermo Fisher Scientific (1 mentions)
Pa5 109888, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 2000 transfection reagent, by Thermo Fisher Scientific (1 mentions)
Lsm780 confocal laser scanning microscope, by Zeiss (1 mentions)
Bromodeoxyuridine (brdu), by Merck Group (1 mentions)
Bromodeoxyuridine (brdu), by Abcam (1 mentions)
Ab6326, by Abcam (1 mentions)
10 protocols using ibitreat 8 well μ slides
1
Fluorescent Imaging of Cell Lines
2
BODIPY Staining and Confocal Imaging
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Cells for BODIPY staining and subsequent confocal imaging were cultured on ibiTreat 8-well μ-Slides (Ibidi GmbH, Planegg/Martinsried, Germany). Cells were fixed in a 2% formalin solution diluted in PBS 1X at room temperature (RT) for 15 min. Subsequently, after three washes in PBS 1X, cells were incubated in a solution of BODIPY 493/503 in PBS 1X to fluorescently label lipid droplets. The incubation was performed at RT in dark for 45 min. After the incubation, the slides were washed in PBS 1X three times and then mounted with a DAPI-containing mounting medium. Fluorescent images were obtained using a Leica SP8 confocal microscope (Leica Microsystems Srl, Milan, Italy) equipped with LAS X 3.1.5.16308 software. Slides were observed with HC PL APO CS2 40X/1.10 WATER or HCX PL APO lambda blue 63X/1.40 OIL objective lenses. DAPI fluorescence was detected by diode 405 laser (410/480 nm), while the fluorescence of BODIPY was detected by white light laser (503/588 nm). The images were acquired by a photomultiplier tube (PMT), which allowed point-by-point scanning of the region of interest (ROI) with the selected laser and produced 1024 × 1024 px images.
3
Quantitative Immunofluorescence Analysis
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For immunofluorescence staining, cells were fixed for 10 min with 4% EM grade formaldehyde. After 5 min washing with PBS, samples were permeabilized for 10 min with 0.5% TX-100 in PBS. Unspecific binding was blocked by 30 min incubation with 5% BSA (Sigma) at RT. Cells were then incubated overnight (4 °C) with the primary antibody for filamin A diluted according to the manufacturer’s instructions (1:400, Thermo Fisher, MA5-11705). After 3 x 10 min washing with PBS, samples were incubated with secondary antibodies (1:500, AF488 goat-anti-mouse AB_2534069), rhodamine phalloidin (1:300, Sigma-Aldrich) and DAPI (0.5 μg/ml, Sigma-Aldrich) for 1 hour at RT, washed again 3 x 10 min with PBS. All stainings were performed in ibiTreat 8 well μ-slides (ibidi GmbH) coated with fibronectin (Corning). Total fluorescence intensities and nuclear shape factors were quantified using ImageJ v1.52. Z-plane scaled 3D stacks were rendered using the Leica LAS X software platform.
4
Modulation of Cell Differentiation Pathways via miRNA Mimics
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Cells were plated on 12-well cell culture plates at a density of 60,000 cells per well, or on ibiTreat 8-well μ-slides (ibidi GmbH, Munich district, Germany), at a density of 10,000 cells per well and maintained until 80% confluence. The cells were treated with mimics for a negative control, miR-16-5p, or miR-27b-3p (4.8 pmol for 12-well plates and 1.2 pmol for ibiTreat 8-well μ-slides) with a combination of overexpression vectors [100 ng (12-well plates) or 25 ng (ibiTreat 8-well μ-slides)] using Lipofectamine 3000 transfection reagent (Thermo Fisher Scientific), according to the manufacturer’s protocol, which was followed by treatment with Eda (Antibodies-online Inc., ABIN3291185), Relt (Antibodies-online Inc., ABIN4054001), or Smad3 (Antibodies-online Inc., ABIN3809504) for the miR-16-5p mimic, or Bmp2 (Antibodies-online Inc., ABIN4045152), Pax9 (Antibodies-online Inc., ABIN4216431), or Slc24a4 (Addgene, 75208) for the miR-27b-3p mimic (n = 6 per group). After 24 h of transfection, the medium was switched to differentiation medium for 2 days.
5
Regulation of AMELX Expression
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The cells were plated onto ibiTreat 8-well μ-slides (ibidi GmbH, Munich district, Germany) at a density of 10,000/chamber and maintained until 80% confluency. The cells were then treated with mimics for miR-16-5p, miR-27b-3p, or a negative control, using Lipofectamine RNAiMAX transfection reagent (4.8 pmol of mimic with 0.48 µL of transfection reagent in 200 µL of differentiation medium) (n = 4 per group). After 24 h of treatment, the medium was replaced with differentiation medium for 2 days. AMELX expression was detected with anti-AMELX rabbit polyclonal antibody (ab153915, Abcam, 1:250), as previously described (Yoshioka et al., 2021b (link)). Immunofluorescent images were captured with a confocal microscope (Ti-E, Nikon United States).
6
Mitochondrial Imaging in SGBS Cells
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SGBS cells cultured on ibiTreat 8-well μ-slides (Ibidi GmbH, Germany) were incubated at 37°C with 100 nM MitoTracker® Orange CMTMRos (Thermo Fisher Scientific, USA) for 30 minutes. The stained cells were washed with PBS 1X and fixed with 2% formalin at RT for 15 minutes. After fixation, cells were rinsed three times with PBS 1X, mounted in DAPI-containing mounting medium (Cayman Chemical Company, USA), and imaged using a Leica SP8 confocal microscope (Leica Microsystems, Germany) and LAS X 3.1.5.16308 software. Slides were viewed with the HCX PL APO lambda blue 63x/1.40 OIL objective. DAPI fluorescence was detected with a 405 diode laser (410/480 nm), while MitoTracker® fluorescence was detected with a white light laser (550/605 nm). Images were acquired using a photomultiplier tube (PMT) that allowed point-by-point scanning of the region of interest (ROI) with the selected laser and produced images with a resolution of 1024 x 1024 px.
7
Regulation of KLK4 and MMP20 by miRNA mimics
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The cells were plated onto ibiTreat 8-well μ-slides (ibidi GmbH, Munich district, Germany) at a density of 5000/chamber and cultured until 80% confluency. Then, cells were treated with mimic for miR-3195, miR-1306-5p, miR-3914, or control using Lipofectamine RNAiMAX transfection reagent (4.8 pmol of mimic with 0.48 µL of transfection reagent in 200 µL of keratinocyte-SFM medium). After 24 h, the cells were cultured with differentiation medium for 72 h. Immunofluorescence analysis was performed, as previously described [55 (link)], using rabbit polyclonal antibodies against KLK4 (PA5-109888, Thermo Fisher Scientific, 1:200) and MMP20 (55467-1-AP, Proteintech, 1:250). Images were taken with a confocal microscope (Ti-E, Nikon USA, Melville, NY, USA).
8
Visualizing Antiviral Protein Localization
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Human embryonic kidney 293T cells (20 000 cells per well) were seeded onto ibiTreat 8-well μ-slides (Ibidi) and co-transfected with A3G-YFP, A3F-mCH, A3D-mCH and A3H-mCH expression plasmids using the Lipofectamine™2000 transfection reagent (Invitrogen). The cells were fixed 18-h post-transfection with 4.0% paraformaldehyde and cellular DNA was counterstained with DAPI (Sigma). The transfected cells were visualized using a LSM780 laser-scanning confocal microscope (Zeiss).
9
Lipid Droplet Visualization by BODIPY Staining
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Cells for BODIPY™ staining and subsequent confocal imaging were cultured on ibiTreat 8-well μ-slides (Ibidi GmbH, Planegg/Martinsried, Germany). Cells were fixed in a 2% formalin solution diluted in PBS 1X at room temperature (RT) for 15 minutes. Subsequently, after washing three times in PBS 1X, the cells were incubated in a solution of BODIPY™ 493/503 in PBS 1X to fluorescently label the lipid droplets. Incubation was performed for 45 minutes in the dark at RT. The slides were then washed three times in PBS 1X.
Fluorescence images were acquired using a Leica SP8 confocal microscope (Leica Microsystems Srl, Milan, Italy) and LAS X 3.1.5.16308 software. Slides were viewed with the HCX PL APO lambda blue 63x/1.40 OIL objective. DAPI fluorescence was detected with a 405 diode laser (410/480 nm), while BODIPY fluorescence was detected with a white light laser (503/588 nm). Images were acquired using a photomultiplier tube (PMT) that allowed point scanning of the region of interest (ROI) with the selected laser and produced 1024 × 1024 px images.
Fluorescence images were acquired using a Leica SP8 confocal microscope (Leica Microsystems Srl, Milan, Italy) and LAS X 3.1.5.16308 software. Slides were viewed with the HCX PL APO lambda blue 63x/1.40 OIL objective. DAPI fluorescence was detected with a 405 diode laser (410/480 nm), while BODIPY fluorescence was detected with a white light laser (503/588 nm). Images were acquired using a photomultiplier tube (PMT) that allowed point scanning of the region of interest (ROI) with the selected laser and produced 1024 × 1024 px images.
10
miRNA Regulation of Neural Differentiation
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mHAT9d cells were plated onto ibiTreat 8-well μ-slides (ibidi GmbH, Munich district, Germany) at a density of 10,000/chamber and cultured until 80% confluence. Cells were then treated with a mimic for miR-16-5p, miR-27b-3p, or control using Lipofectamine RNAiMAX transfection reagent (4.8 pmol of mimic with 0.48 µL of transfection reagent in 200 µL of proliferation medium). After 24 h of transfection, the cells were cultured under differentiation medium for 48 h. In addition, cells were treated with 100 μg/ml BrdU (Sigma Aldrich) for 1 h at day 2 of differentiation (n = 6 per group) and visualized with a rat monoclonal antibody against BrdU (ab6326; Abcam, 1:1,000), as previously described (Yoshioka et al., 2021a (link)). BrdU-positive cells were quantified using images from six independent experiments.
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