MDCK cells were trypsinized to a single cell suspension at 4 × 104 cells/ml in complete medium containing 2% Matrigel (BD Biosciences). Cell-medium-Matrigel suspensions (250 µl) were plated into µ-Slide 8 well ibiTreat chambers (Ibidi, 80826), precoated with 15 µl of Matrigel (10 mg/ml). For long cultures, medium was replaced every 2 days. Cells were grown at different time points before fixation in 4%paraformaldehyde (PFA).
Ibitreat chambers
Manufactured by Ibidi
Sourced in Germany
IbiTreat chambers are laboratory equipment designed for cell culture applications. They provide a controlled environment for the incubation and treatment of cell samples. The chambers maintain temperature, humidity, and gas levels to support optimal cell growth and experimentation.
Automatically generated - may contain errors
Lab products found in correlation
Matrigel, by BD (2 mentions)
Matrigel, by Ibidi (2 mentions)
Lsm 880, by Zeiss (2 mentions)
Collagen 1, by Ibidi (1 mentions)
Collagen 1, by Corning (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Envision system, by Agilent Technologies (1 mentions)
12 well glass bottom plate, by Cellvis (1 mentions)
Biostation im q time lapse imaging system, by Nikon (1 mentions)
4 well culture plates, by Thermo Fisher Scientific (1 mentions)
Rat tail collagen type 1, by BD (1 mentions)
Penicillin streptomycin, by Merck Group (1 mentions)
Texas red phalloidin, by Thermo Fisher Scientific (1 mentions)
Anti zo 1, by Thermo Fisher Scientific (1 mentions)
Protein block reagent, by Agilent Technologies (1 mentions)
Zen software, by Zeiss (1 mentions)
Atn 161, by Bio-Techne (1 mentions)
Mouse monoclonal anti vinculin, by Abcam (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Avantor (1 mentions)
Triton x 100, by Merck Group (1 mentions)
9 protocols using ibitreat chambers
1
MDCK 3D Culture with Matrigel
2
Caco-2 Cyst Formation in 3D Culture
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Caco-2 cells were trypsinized to a single cell suspension at 6 × 104 cells/ml and mixed with 20 mM HEPES pH7.5, 1 mg/ml Collagen I (Corning®), 50% Matrigel (BD Biosciences). Final volume was adjusted with complete medium containing. Cell-Matrix-medium suspensions (250 µl) were plated into µ-Slide 8 well ibiTreat chambers (Ibidi, 80826), and left in the incubator at 37 °C for at least 30 min to allow the mix to solidify through polymerization of Collagen I and Matrigel. The solidified mixture was then supplemented with 250 µl of complete medium. Cells were grown at different time points before fixation in 2%paraformaldehyde (PFA). For long cultures, medium was replaced every 2 days. To speed-up lumen formation in mature cysts, Cholera Toxin (0.1 µg/ml;) was added at day 6 and cysts culture fixed at day 7.
3
Immunohistochemistry and Immunofluorescence Staining Protocol
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Formalin-fixed skin samples were processed for paraffin wax embedding. Endogenous peroxidase activity in sections was quenched by incubation with 1% H2O2 for 30 min and antigen access was recovered by heating in citrate buffer pH 6. Sections were incubated with primary antibodies for 90 min at room temperature (Table S2 ) after blocking in 10% goat serum. Sections were washed in water and then incubated in secondary antibody using the EnVision system (Dako) for 30 min at room temperature. Peroxidase activity was detected with diaminobenzidine tetrahydrochloride (DAB) substrate (Dako). Sections were counterstained with hematoxylin.
For immunofluorescence, frozen sections or cells grown on ibiTreat chambers (Ibidi) were fixed in methanol/acetone (1:1) at –20°C for 10 min and blocked in 10% goat serum as above. Sections or cells were incubated with primary antibodies for 60 min (Table S2 ), followed by incubation with secondary antibody coupled to Alexa Fluor 488 (Molecular Probes; Life Technologies) and counterstained with DAPI.
For immunofluorescence, frozen sections or cells grown on ibiTreat chambers (Ibidi) were fixed in methanol/acetone (1:1) at –20°C for 10 min and blocked in 10% goat serum as above. Sections or cells were incubated with primary antibodies for 60 min (
4
Time-Lapse Imaging of Mitosis in HeLa Cells
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HeLa‐H2B/tub cells 30 expressing H2B‐mCherry and EGFP‐α‐tubulin were kindly provided by Jan Ellenberg (EMBL Heidelberg, Germany) and used for time‐lapse microscopy as described previously 67 . Briefly, cells were plated on μ‐slide 8‐well ibiTreat chambers (Ibidi) and imaged at 5‐min intervals for 48 h on a Nikon BioStation IM‐Q Time Lapse Imaging System using a 20×/0.8 NA air objective, a 1.3 megapixel cooled monochrome camera (Nikon), and Nikon software for image acquisition. Alternatively, HeLa‐H2B/tub cells were imaged at 15‐min intervals on a DeltaVision deconvolution microscope using a 20×/0.75 air objective.
HeLadox‐YFP‐TIARr cells were plated on a 12‐well glass bottom plate (Cellvis) and imaged at 15‐min intervals for 12 h on a Nikon Ti‐E microscope with an integrated perfect focus system using a 40x/0.95 NA air objective, a 4.2 megapixel cooled monochrome sCMOS pco.pge camera, and NIS elements JOBS software for image acquisition. All cells were kept in 10% FBS/DMEM at 37°C and 5% CO2 during imaging. Image processing was performed using ImageJ software (NIH,http://rsbweb.nih.gov/ij/ ).
HeLadox‐YFP‐TIARr cells were plated on a 12‐well glass bottom plate (Cellvis) and imaged at 15‐min intervals for 12 h on a Nikon Ti‐E microscope with an integrated perfect focus system using a 40x/0.95 NA air objective, a 4.2 megapixel cooled monochrome sCMOS pco.pge camera, and NIS elements JOBS software for image acquisition. All cells were kept in 10% FBS/DMEM at 37°C and 5% CO2 during imaging. Image processing was performed using ImageJ software (NIH,
5
Immunofluorescence Imaging of Megakaryocytes
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For immunofluorescence, MKs in suspension were fixed and permeabilized in one step for 30 min in PBS with 3.7% formaldehyde and 0.05% Triton X-100. Samples were saturated in 1% fatty acid free BSA in PBS. Incubation with antibodies, fluorescent secondary antibodies, AlexaFluor-labelled phalloidin or DAPI was performed for 1 h at RT. For 3D imaging, cells were kept in μ-slide ibiTreat chambers (Ibidi) in PBS. Confocal images were captured with a LSM780 operated with Zen software using a × 63, 1.4 NA Plan Apochromatic objective lens (Carl Zeiss). Profiling of fluorescence intensity was done with ImageJ (NIH). For 3D-analysis, z-stacks were taken and processed with the Imaris 6.4.2 software (Bitplane AG).
6
Culturing Sympathetic Neurons on Collagen
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Sympathetic neurons were plated on collagen coated culture plates. Surface coating was performed under sterile conditions on the same day as the embryo preparation. Rat tail collagen (type I) (BD Bioscience, USA) was diluted using 0.02 N acetic acid to a concentration of 50 µg/ml. Around 200 µl of diluted collagen solution were pipette in every well and left at RT for 1 h. Wells were then washed 4 × 5 min using 500 µl PBS and left under the sterile hood until neurons were plated.
Neurons were plated on either 4-well culture plates (Nunc, Denmark, EU) or 15 µ-Slide 8 well ibiTreat chambers (Ibidi, Germany, EU) at a final concentration of 20,000 cells per 200 or 500 µl (depending on the well-plates used). One day after plating approximately 2/3 of the N2-Medium were substituted by Medium A (N2-Medium without FCS, 1 % B-27 supplement, 0.24 µg/ml 1-β-arabinofuranosylcytosine (Ara-C) and 0.2 % penicillin/streptomycin (Sigma-Aldrich, Germany, EU). On DIV3, all medium was completely substituted by Medium A. Medium was then changed every 2 days until DIV7.
Neurons were plated on either 4-well culture plates (Nunc, Denmark, EU) or 15 µ-Slide 8 well ibiTreat chambers (Ibidi, Germany, EU) at a final concentration of 20,000 cells per 200 or 500 µl (depending on the well-plates used). One day after plating approximately 2/3 of the N2-Medium were substituted by Medium A (N2-Medium without FCS, 1 % B-27 supplement, 0.24 µg/ml 1-β-arabinofuranosylcytosine (Ara-C) and 0.2 % penicillin/streptomycin (Sigma-Aldrich, Germany, EU). On DIV3, all medium was completely substituted by Medium A. Medium was then changed every 2 days until DIV7.
7
Immunofluorescence Staining of Tight Junction Proteins in HBMEC Cells
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HBMEC cells were grown on µslides 8-well Ibitreat chambers (Ibidi) for 10 days and the medium was changed every other day. The medium was removed, and cells were fixed with 4% formaldehyde (EMS) for 20 min at room temperature, washed 3 times with sterile PBS (pH 7.4), incubated with glycine (0.1 M), and permeabilized with Triton X-100 (0.02%) for five minutes at room temperature. Cells were blocked with Protein-Block reagent (Dako, X090930-2) for 30 min and stained for one hour with rabbit polyclonal anti-ZO1 (1:50 dilution; clone 617,300; Thermo Fisher Scientific), and Texas Red phalloidin (1:1000 dilution; Thermo Fisher Scientific). Cells were then washed 3 times with sterile PBS (pH 7.4) and incubated with anti-rabbit IgG AlexaFluor 488-linked antibody (green signal, 1:1000; Thermo Fisher Scientific) for one hour in the dark. Antibody dilutions were performed using antibody diluent (Dako, S302281-2). HBMECs were washed 3 times with PBS, co-stained with Hoechst for 15 min. Cell monolayers were imaged using a Zeiss LSM 880 laser scanning confocal microscope with 40 × and 63 × oil objectives. Images were processed using the Zen software (Zeiss).
8
Integrin Inhibition and Shear Stress
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HUAECs were seeded in µ-slide I0.4 Luer ibiTreat chambers (80176, Ibidi) and unidirectional laminar or oscillatory flow was applied as described above. After cells were adapted to laminar flow for 24 h, confluent monolayers were treated with 50 μmol/L of the α5β1 integrin inhibitor ATN-161 (Tocris, 6058) or with 1 μmol/L of the αv integrin inhibitor S247 (Tocris, 5870), for 10 min and shear stress-induced signaling was assessed.
9
Cellular Morphology and Cytoskeleton Analysis
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Qualitative and semi-quantitative analysis of cell morphology was performed on nHA-pGFP, PEI-pGFP and RALA-pGFP transfected cells. Cells were imaged at day 1 and 3 after transfection using an inverted epifluorescent microscope (Olympus IX83, Germany). Cell morphology was assessed semiquantitatively through the calculation of the cell surface area, aspect ratio and circularity using ImageJ software, n = 4 and 6 pictures per sample.
Analysis of actin cytoskeleton and focal adhesion points on vector-pLUC transfected cells were performed by actin and vinculin immunofluorescent staining. At day 1 and 3 after transfection, MSCs previously seeded on µ-slide 4 well IBItreat chambers (IBIDI, Germany), were fixed in 4% paraformaldehyde (PFA) (Sigma-Aldrich, Ireland) for 15 min at room temperature (RT). Samples were first blocked with 5% bovine serum albumin (BSA) and incubated overnight with vinculin primary antibody (AB) (1:500 α mouse monoclonal anti-vinculin) (Abcam, ab18058, Ireland) at 4 o C. After incubation, samples were permeabilised with 0.5% Triton X-100 (Sigma-Aldrich, Ireland) and incubated with secondary AB (1:250 α mouse Alexa Fluor 488 IgG) (Biosciences, Ireland) and stained with rhodamine phalloidin (VWR, Ireland) for 1 h at RT. Cell nuclei were stained with DAPI (VWR, Ireland) for 10 min at RT, washed in PBS and were imaged using confocal microscopy (Leica SP8, Ireland).
Analysis of actin cytoskeleton and focal adhesion points on vector-pLUC transfected cells were performed by actin and vinculin immunofluorescent staining. At day 1 and 3 after transfection, MSCs previously seeded on µ-slide 4 well IBItreat chambers (IBIDI, Germany), were fixed in 4% paraformaldehyde (PFA) (Sigma-Aldrich, Ireland) for 15 min at room temperature (RT). Samples were first blocked with 5% bovine serum albumin (BSA) and incubated overnight with vinculin primary antibody (AB) (1:500 α mouse monoclonal anti-vinculin) (Abcam, ab18058, Ireland) at 4 o C. After incubation, samples were permeabilised with 0.5% Triton X-100 (Sigma-Aldrich, Ireland) and incubated with secondary AB (1:250 α mouse Alexa Fluor 488 IgG) (Biosciences, Ireland) and stained with rhodamine phalloidin (VWR, Ireland) for 1 h at RT. Cell nuclei were stained with DAPI (VWR, Ireland) for 10 min at RT, washed in PBS and were imaged using confocal microscopy (Leica SP8, Ireland).
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