Cells were seeded on titanium discs (n = 648) at a concentration of 104 cells/well in a 48-well plate (BD, Milan Italy) and then kept in growth condition. After 20 min, 24 h and 72 h (T0, T1, T2), the titanium specimens were washed in Phosphate Buffer Saline (PBS) and then the cells were fixed with 4% paraformaldehyde (PFA) in PBS for 15 min. After two washes with PBS, cells were permeabilized with 0.1% Triton X-100 (Sigma-Aldrich) in PBS. Following the manufacturer’s protocol, cells were stained with Rodhamine-Phalloidin (Life Technologies) and 1 uM 4′,6-diamidino-2-phenylindole (Dapi, Life Technologies) to respectively detect the cytoskeleton and the nuclei [45 (link),46 (link)]. Image acquisition was made recurring to a Nikon Eclipse Ti-E microscope with 40X objective (Plan Fluor Nikon) [47 (link)]. Image analysis [48 (link),49 (link),50 (link)] was performed using ImageJ software (ImageJ, U.S. National Institutes of Health, Bethesda, MA, USA, http://imagej.nih.gov/ij/ ).
48 well plate
Manufactured by BD
Sourced in United States, Italy, Canada
The 48-well plates are a laboratory equipment designed for a variety of cell culture and experimental applications. The plates consist of 48 individual wells, each capable of containing a small volume of liquid sample or cell culture medium. The plates are typically made of durable, non-toxic materials to ensure compatibility with a wide range of experimental protocols.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (6 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (3 mentions)
Eclipse t e microscope, by Nikon (2 mentions)
Triton x 100, by Merck Group (2 mentions)
Anti cd45 pe cy7, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 700 anti cd11c, by Thermo Fisher Scientific (2 mentions)
Pacific blue anti mhcii, by BioLegend (2 mentions)
Rpmi 1640, by Merck Group (2 mentions)
L glutamine, by Thermo Fisher Scientific (2 mentions)
Matrigel, by BD (2 mentions)
Mabs cd3 ucht1, by BD (2 mentions)
Soluble cd28 cd28.2, by BD (2 mentions)
Intracellular ifn γ 4s b3, by BD (2 mentions)
T cell isolation kit, by STEMCELL (2 mentions)
Leukocyte activation cocktail, by BD (2 mentions)
Synergy ht, by Agilent Technologies (2 mentions)
Celltiter 96 aqueous one solution cell proliferation assay, by Promega (2 mentions)
Alphalisas, by PerkinElmer (2 mentions)
Quantikine elisa, by R&D Systems (2 mentions)
Rodhamine phalloidin, by Thermo Fisher Scientific (1 mentions)
55 protocols using 48 well plate
1
Cellular Cytoskeletal Visualization on Titanium
2
Gliadin Peptide Exposure in NOD/DQ8 Mice
3
Cloning of Chromosomal Flanking Regions
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DNA was isolated from the G418-resistant, polyclonal HT-1080/LHSNO cells present in ten 10 cm dishes and the G418-resistant, polyclonal AAV-targeted HT-1080/LHSN63Δ53O cells present in twenty 10 cm dishes. The shuttle vector target sites, along with flanking chromosomal DNA, were rescued as bacterial plasmids as described previously 65 (link), except that three pairs of compatible, cohesive restriction enzymes were used: EcoRI/MfeI; BsrGI/BsiWI; and PciI/BspHI. Transformed bacteria were selected on agar containing 50 μg kanamycin/ml. Nine thousand colonies from bacterial transformations of HT-1080/LHSNO DNA were inoculated into individual wells of a 48-well plate (BD Biosciences), each containing 500 μl LB medium with 50 μg kanamycin/ml, to avoid overgrowth of individual clones. Nine thousand colonies from transformations of AAV-targeted HT-1080/LHSN63Δ53O DNA were inoculated in an identical way. Cultures were grown overnight at 37°C, at which point the colonies were pooled, and plasmid DNA was purified for sequencing.
4
Cloning of Chromosomal Flanking Regions
5
Cytoskeleton Analysis of Cell Morphology
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Cells were seeded on the samples at a concentration of 7000 cells/sample in a 48-well plate (BD) and then kept in growth conditions. After 1 and 24 h, the specimens were washed in PBS, before fixing the cells with 4% paraformaldehyde in PBS for 10 min. After being washed with PBS, cells were permeabilized with 0.1% Triton X-100 (Sigma-Aldrich, Milano, Italy) in PBS. Cells were stained with Alexa 488-Phalloidin (Life Technologies, Milano, Italy) to detect the cytoskeleton. Images were acquired with a Nikon Eclipse Ti-E microscope using different objectives: Nikon Plan 10X/0.10; Nikon Plan Fluor 40X/0.75; and Nikon Plan Apo VC 60X/1.40 (Nikon Instruments, Amsterdam, The Netherlands).
A directionality analysis was performed using an automated software developed in our laboratory called MORPHEUS. The tool was used according to the workflow reported in the literature [32 (link)].
A directionality analysis was performed using an automated software developed in our laboratory called MORPHEUS. The tool was used according to the workflow reported in the literature [32 (link)].
6
Generation of Mature Dendritic Cells
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Peripheral blood mononuclear cells were isolated from peripheral blood from healthy adult volunteer donors by density gradient centrifugation on Lymphoprep centrifuged at 1500 rpm for 20 min. Monocytes were recovered as much as possible at the interface and washed three times in PBS containing 2% autologous plasma and 2 mmol/ml EDTA. The CD14+ monocytes were purified from the suspension using the Human CD14 Positive selection kit and resuspended with a concentration of 1 × 106 cells/ml in RPMI medium with 10% FBS. The cell suspension was divided into 0.5 ml each and cultured in the 48-well plate (BD Pharmingen) in the presence of GM-CSF (50 ng/ml), IL-4 (10 ng/ml), and IFN-β (5 × 103 U/ml) at 37oC in a 5% CO2 humidified incubator for 48 hours. Then the IL-1β (10 ng/ml) and poly I:C (20 µg/ml) were added as maturation stimuli, culturing for 24 hours to promote maturation. The G4-DCs were cultured in the presence of GM-CSF (50 ng/ml) and IL-4 (10 ng/ml) in a 5% CO2 humidified incubator for 5 days. Then the IL-1β (10 ng/ml) and poly I:C (20 µg/ml) were added, culturing for 48 hours to promote maturation. The G4B-DCs and G4-DCs were harvested at the same time.
7
Quantifying Titanium Cell Adhesion
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Cell adhesion was evaluated on 648 titanium samples using a 48-well plate (BD, Milan, Italy). Cells were detached using trypsin for 3 min, carefully counted, and seeded at 3 × 103 cells/well in 1 mL of growth medium on the different samples. The 48-well plates were kept at 37 °C, 0.5% CO2 for 20 min, 24 h and 72 h. Before and after fixation in 4% paraformaldehyde in PBS for 15 min at room temperature, cells were washed two times with PBS and then stained with 1 μM DAPI (Molecular Probes, Eugene, CA, USA) for 15′ at 37 °C to detect cell nuclei. Samples were analyzed using a Nikon Eclipse T-E microscope with a 4X objective. Cell nuclei were then counted by using ImageJ (NIH) software with the tool “Analyze particles” [51 (link),52 (link)].
8
Clofarabine Modulates Neural Stem Cell Differentiation
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In all experiments NSCs were cultured in serum-free DMEM/F12 for 12 h, unless otherwise stated. For NSC differentiation assays, 100-µm-diameter neurospheres were collected by centrifugation (1,000 × g for 10 min at 4°C) and 10–15 neurospheres were seeded onto a 48-well plate (BD Biosciences) in stem cell differentiation induction medium [DMEM/F12 containing 4% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.), 2% B27 and 2% N2]. Following 12-h culture, differentiating NSCs were treated with Clo (5 or 10 µM) or DMSO (control) at 37°C and differentiation was evaluated after culture in stem cell differentiation induction medium after 7 days of treatment.
9
In Vitro Osteoblast Cell Adhesion
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Among the different osteoblast and pre-osteoblast cell model available 24 -26 , the widely diffused pre-osteoblastic murine cell line MC3T3-E1 (ECACC, Salisbury, UK) was used to characterize the biological response in vitro 24 . Cells were maintained in Alpha MEM supplemented with 10% fetal bovine serum (Life Technologies, Milan, Italy), 100 U/ml penicillin, 100 mg/ml streptomycin, were passaged at subconfluency to prevent contact inhibition and were kept under a humidified atmosphere of 5% CO2 in air, at 37°C. Cell adhesion was evaluated on titanium samples using a 48-well plate (BD, Milan Italy). Cells were detached using trypsin for 3 minutes, carefully counted and seeded at 3 × 10 3 cells/well in 100 μl of growth medium on the different samples. The 48-well plates were kept at 37 °C, 0.5 % CO2 for 20 min. Before and after fixation in 4% paraformaldehyde in PBS for 15 min at room temperature, cells were washed two times with PBS and then stained with 1μM DAPI (Molecular Probes, Eugene, California, USA) for 15' at 37°C to stain cell nuclei. Images were acquired using a Nikon Eclipse T-E microscope 27, 28 . Cell nuclei were counted using the 'Analyze particles' tool of ImageJ software (ImageJ, U. S. National Institutes of Health, Bethesda, Maryland, USA, http://imagej.nih.gov/ij/) 29, 30 .
10
Bone Marrow-Derived Dendritic Cell Assay
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Bone marrow cells from C57BL/6 and P2X7R−/− mice were cultured in BMDC culture media containing RPMI-1640 (Sigma-Aldrich) with 10% FBS (Thermo Fisher Scientific), 1% penicillin/streptomycin, 1% l -glutamine, 50 μM β-mercaptoethanol and 4% granulocyte–macrophage colony-stimulating factor. After 6 days of culture, the phenotype of BMDCs was confirmed by staining with PE-Cy7-anti-CD45, Alexa Fluor 700-anti-CD11c (eBioscience) and Pacific Blue-anti-MHCII (BioLegend) antibodies, and the purity of MHCII+CD45+CD11c+ BMDCs was >90%. Splenocytes (106 cells per ml) from C57BL/6 OT-II mice were cocultured with OVA-pulsed BMDCs from WT or P2X7R−/− mice at a ratio of 10:1 in a 48-well plate (BD Falcon) for 72 h.
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