To bioprint BoneMA in various geometrical shapes, specific print patterns were designed using CAD (Fusion 360, AutoDesk) and converted into image slices with the accompanying 3D printing software (Print Studio, Autodesk). The print patterns were designed arbitrarily with star, square, triangle, and rhombus shapes, as well as flower, spiral, concentric circles, and the OHSU logo. Shapes were printed with length and width dimensions that ranged from 600 μm (square) to 2.5 mm (OHSU logo) with a thickness of 450 μm, under printing exposures of 25 seconds. For bioprinting of the said geometries, the bioink was formulated by mixing HMSCs with the BoneMA (5% (w/v)) pre-polymer containing LAP (0.15% (w/v)) at a concentration of 5 × 105 cells/ml. The HMSC-laden bioprinted BoneMA was fixed with 4% (v/v) paraformaldehyde and permeabilized using 0.1% (v/v) Triton X-100. Next, the samples were treated with 1.5% (w/v) bovine serum albumin (Sigma Aldrich) in DPBS for 1h, followed by Image-iT FX signal enhancer (Invitrogen, CA) for 30 min, after which they were immunostained with ActinGreen™ 488 ReadyProbes™ (Invitrogen).
Actingreen 488 readyprobe
Manufactured by Thermo Fisher Scientific
Sourced in United States
ActinGreen™ 488 ReadyProbes® is a fluorescent probe designed for the detection and visualization of actin filaments in fixed cells. It provides a simple and convenient method for labeling actin with the green-fluorescent dye Alexa Fluor® 488.
Automatically generated - may contain errors
Lab products found in correlation
Nucblue live readyprobe, by Thermo Fisher Scientific (4 mentions)
Evos fl cell imaging system, by Thermo Fisher Scientific (3 mentions)
Actinred 555 readyprobe, by Thermo Fisher Scientific (3 mentions)
Infinite m200, by Tecan (2 mentions)
A21203, by Thermo Fisher Scientific (2 mentions)
Fusion 360, by Autodesk (1 mentions)
Image it fx signal enhancer, by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Print studio, by Autodesk (1 mentions)
Image it fix perm kit, by Thermo Fisher Scientific (1 mentions)
Prolong diamond antifade mountant, by Thermo Fisher Scientific (1 mentions)
Formaldehyde, by Merck Group (1 mentions)
Evos fl, by Thermo Fisher Scientific (1 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (1 mentions)
Tcs sp8 confocal microscope system, by Leica (1 mentions)
Paraformaldehyde (pfa), by Mallinckrodt (1 mentions)
Axio observer z1 lsm800, by Zeiss (1 mentions)
C plan apo 63x 1.4 oil dic objective, by Zeiss (1 mentions)
Zen 2.3 image analysis software package, by Zeiss (1 mentions)
F actin, by Thermo Fisher Scientific (1 mentions)
26 protocols using actingreen 488 readyprobe
1
Bioprinting BoneMA Geometrical Shapes
2
Fixation, Permeabilization, and Actin Staining
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Cell cultures were fixed with 4% paraformaldehyde in PBS for 10 min and then washed in PBS thrice. Following this, they were permeabilized with 0.5% Triton X-100 for 10 min and washed in PBS thrice. After the cells were fixed and permeabilized, they were incubated in a blocking solution of 3% bovine serum albumin in PBS for 60 min, following which ActinGreen 488 ReadyProbes (2 drops/ml; Invitrogen) was added to the blocking solution. The solution was incubated for another 30 min with the actin stain before washing and imaging in PBS.
3
Aluminum Nanoparticle Exposure Analysis
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All cell types were seeded and incubated into one of 4 wells of a chamber slide (Lab-Tek II, Rochester, New York) for 24 h to allow adhesion and acclimation. Cells were treated with 1 ppm aluminum nanoparticle suspension for 24 h. The cells were washed three times prior to fixation, permeabilized as described in the Image-it Fix-Perm kit (Molecular Probes, Eugene, Oregon, USA), and stained with ActinGreen 488 ReadyProbes and NucBlue Live ReadyProbes (Invitrogen). Each well was washed with phosphate buffered saline (PBS) three more times and fixed with two drops of ProLong Diamond Anti-fade Mountant (Molecular Probes). A glass cover slip was carefully placed on the slide and set for 24 h. Fluorescent images of the cells were first used to focus on the sample before switching to hyperspectral imaging (CytoViva Inc., Auburn, Alabama, USA) with the accompanying ENVI software (Advanced Scientific Camera Control Version 1.0).
4
Visualizing Differentiated Muscle Cells
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In vitro differentiated human muscle stem cells were fixated with 4% Formaldehyde (Sigma) and then permeabilized with 0.5% Triton X-100. Cells were then incubated with ActinGreen 488 ReadyProbes and a nuclear counterstain was performed with Nucblue Fixed Cell stain ReadyProbes (Molecular probes). Fluorescence microscopy was performed with an EVOS FL (Thermo Fisher).
5
Fluorescent Actin Staining Procedure
6
Fluorescent Staining of Actin Cytoskeleton
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MDMs or iMacs were washed in PBS and fixed in 4% cold paraformaldehyde for 10 min. The cells were washed again in PBS and permeabilized in 0.1% Triton X‐100. The cells were washed in PBS again and then stained with either ActinGreen™ 488 ReadyProbes® or ActinRed™ 555 ReadyProbes® reagent (Molecular Probes) for 30 min followed by 2 more washes in PBS. The cells were then imaged with the EVOS™ FL Cell Imaging System (Invitrogen).
7
Measuring F-actin in HT-29 cells
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Prior to the cAMP measurement, HT‐29 cells were seeded in a black flat‐bottom 96‐well plate with 5 × 104 cells per well and incubated for 48 hr in DMEM with 10% FBS and an antibiotic mix at 37°C in 5% CO2. Cells were then treated with TAs and/or antagonists for 3 hr before F‐actin measurement. For the combination of TAs and antagonists, the antagonists were added 30 min prior to addition of the TAs. For treatment with bacteria, we used WT ED99, ΔsadA, and a complemented mutant at MOI of 30 and AAAs, L‐DOPA, and 5‐HTP separately at a concentration of 100 μg ml−1 and incubated the cells for 3 hr at 37°C in 5% CO2. Lysostaphin was then added to the cell cultures, and cells were incubated further for 30 min. The treated cells were washed with DPBS, permeabilised with 0.1% (v/v) Triton X‐100, stained with ActinGreen™ 488 ReadyProbes® (Thermo Fischer) for 30 min and washed again three times with DPBS, and the relative fluorescence intensity was measured at 495 nm for the excitation and 518 nm for the emission using a Tecan Infinite M200.
8
Immunocytochemistry of MGE Progenitors on Matrigel
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MGE progenitors and/or neurons plated on Matrigel-coated glass coverslips or 8-well glass chambers (Thermo Fisher) were fixed with 4% paraformaldehyde (Mallinckrodt Baker) for 15 min, permeabilized with 0.1% Triton ×100 (Mallinckrodt Baker) for 10 min and blocked with blocking buffer (5% BSA in PBS) for 1 h at room temperature (RT). Cells were incubated overnight at 4 °C with primary antibodies and for 1 h at RT with secondary antibodies (listed in Supplementary Table S2 ). Both primary and secondary antibodies were diluted in a blocking buffer. Actin was visualized with ActinGreen 488 ReadyProbes (Thermo Fisher Scientific). Slides/coverslips were incubated with NucBlue (DAPI) for 5 min at RT and mounted with a ProLong® Gold Antifade reagent (Life Technologies). Confocal images were captured by using Leica TCS SP8 confocal microscope system (63× and/or 20× objective). Images were acquired using LAS X software.
9
Quantifying Actin Cytoskeleton Alterations
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HT‐29 cells were seeded in glass‐bottom cell culture dish plates (Greiner) with 1.5 × 106 cells per plate and incubated for 48 hr in DMEM with 10% FBS and an antibiotic mix at 37°C in 5% CO2. Cells were then treated with either TRY or SER at a concentration of 50 μg ml−1 and incubated for 2 hr at 37°C in 5% CO2. Prior to microscopy, the treated cells were washed with DPBS, fixed with 0.4% paraformaldehyde, permeabilised with 0.1% (v/v) Triton X‐100, stained with ActinGreen™ 488 ReadyProbes® (Thermo Fisher) for 30 min and washed again three times with DPBS. Confocal microscopy was performed using a Zeiss Axio Observer Z1 LSM800 equipped with an Airyscan detector and C Plan‐Apo 63x/1.4 Oil DIC objective (Zeiss). Images were acquired via the ZEN 2.3 image analysis software package (Zeiss). Because the changes in fluorescence intensity cannot be sufficiently distinguished simply by eye, ImageJ software was used to calculate the fluorescence intensity. We calculated the fluorescence intensity by subtracting the background signal of the selected area from the total area signal, as previously described (Gavet & Pines, 2010 ).
10
Aortic Elastin Assessment Protocol
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Latex was injected into the left ventricular apex under low pressure until it was visible in the femoral artery. Animals were then fixed in Formalin (10%) for 24 h before transfer to 70% ethanol for dissection and storage. Aortas were then removed from the animals or dissected in situ for photography prior to paraffinization and sectioning (7 µM). Slides were produced for tissue staining or stained with standard stains including Elastin (Verhoeff-Van Gieson, Thermo Scientific, MI, USA) or F-actin (ActinGreen™ 488 ReadyProbes, ThermoFisher Scientific, USA) for quantitative analysis. Elastin integrity score was rated by blinded observers and graded on an arbitrary scale of 5 (indicating high quality elastic fiber) to 1 (indicating severe Elastin fragmentation). For sequential fluorescence in situ hybridization and immunofluorescence microscopy aortas from human and mice were cryosectioned using OCT standard protocol70 .
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