ActinRed™ 555 ReadyProbes™ reagent, NucBlue™ Fixed Cell Stain ReadyProbes™ reagent, and an Image-it™ Fixation/Permeabilization kit (Life Technologies Corp., Eugene, OR, USA) were employed in this study to investigate the condition of the cellular structure when subjected to the 5-FU loaded film solution. A single round (22 mm) collagen-coated coverslip (BioCoat, Bedford, MA, USA) was placed into each well of a 6-well plate for the cells to adhere to. The seeding density of the cells was 0.3 × 106 per well. The total volume of cells and media was 3 mL for each well. Cells were allowed 48 h within an incubator to reach confluency. Control wells where the cell line was not treated did not contain EPS/5-FU samples. Exposure experiments saw the addition of a 100 µL of EPS/5-FU film-forming solution added to the well. At 12 and 36 h after exposure, cells were immediately fixed using 4% formaldehyde in PBS. Once this was achieved, either the actin filaments or nucleus of the cells were marked for analysis with the utilization of epifluorescent stain. All the acquired images were not manipulated or altered prior to measuring corrected total cell fluorescence (CTCF). Photographs for both stains used the same imaging settings for that respective group. Images were viewed and photographed using an Olympus IX50 inverted fluorescence microscope.
Nucblue fixed cell stain readyprobes reagent
Manufactured by Thermo Fisher Scientific
Sourced in United States
NucBlue Fixed Cell Stain ReadyProbes reagent is a cellular stain that binds to DNA, allowing for the visualization of cell nuclei. The reagent is ready-to-use and provided in a convenient dropper bottle format.
Automatically generated - may contain errors
Lab products found in correlation
Actinred 555 readyprobes reagent, by Thermo Fisher Scientific (3 mentions)
Ix50 inverted fluorescence microscope, by Olympus (2 mentions)
Triton x 100, by Merck Group (2 mentions)
Anti nup62 610497, by BD (2 mentions)
Anti oga hpa036141, by Merck Group (2 mentions)
Anti ha tag alexa fluor 647 conjugate, by Cell Signaling Technology (2 mentions)
Horseradish peroxidase hrp conjugated secondary antibodies, by Rockland Immunochemicals (2 mentions)
Anti o glcnac rl2 ab2739, by Abcam (2 mentions)
Alexa fluor 488 anti rabbit igg, by Thermo Fisher Scientific (2 mentions)
Anti flag f3165, by Merck Group (2 mentions)
Irdye secondary antibody, by LI COR (2 mentions)
Anti β actin sc 47778, by Santa Cruz Biotechnology (2 mentions)
Actingreen 488 readyprobes reagent, by Thermo Fisher Scientific (2 mentions)
Image it fixation permeabilization kit, by Thermo Fisher Scientific (1 mentions)
Blocking solution, by Nacalai Tesque (1 mentions)
Fv10i liv, by Olympus (1 mentions)
Rabbit polyclonal anti ha antibody, by Merck Group (1 mentions)
Mouse monoclonal anti v5 antibody, by Merck Group (1 mentions)
Alexa fluor 594 conjugated goat anti mouse, by Thermo Fisher Scientific (1 mentions)
Optimal cutting temperature compound, by Avantor (1 mentions)
26 protocols using nucblue fixed cell stain readyprobes reagent
1
Evaluating 5-FU Film Effects on Cell Structure
2
Flow Cytometry Cell Sorting Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell sorting and flow cytometric analysis for this project was done on instruments in the Stanford Shared FACS Facility. Flow cytometric analysis was carried out on LSRII- and FACScan-class analyzers (BD Biosciences, San Jose, CA). Sorting was carried out on FACSAriaII-class sorters (BD Biosciences), including one obtained using the NIH S10 Shared Instrument Grant S10RR025518–01. Live cells were discriminated on the basis of DAPI exclusion using the NucBlue Fixed Cell Stain ReadyProbes reagent (Life Technologies). Information on fluorescent protein detection appears in the Supplementary Materials and Methods. All flow cytometric data were analyzed using FlowJo software (Tree Star, Ashland, OR). Information on filter sets used for the detection of each fluorescent protein appears in the Supplementary Materials and Methods.
3
Localization of Overexpressed Proteins in SH-SY5Y Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To confirm the location of APP770-V5 and FNDC5-HA, we examined immunofluorescence staining of SH-SY5Y cells transiently expressing these molecules. We washed prepared cells by PBS and fixed them by 4% paraformaldehyde for 15 min at room temperature. Then, these cells were permeabilized by 0.1% Triron-X and blocked by using Blocking solution (Nacalai Tesque, Japan). We used the mouse monoclonal anti-V5 antibody (1:1000; Sigma) and the rabbit polyclonal anti-HA antibody (1:1000; Sigma) for the primary antibodies to detect APP and FNDC5, and then labeled them by Alexa Fluor 594-conjugated goat anti-mouse (1:2000; Life Technologies, MA, USA) and Alexa Fluor 488-conjugated mouse anti-rabbit (1:2000; Life Technologies), respectively. As the mounting agent, we used NucBlue Fixed Cell Stain ReadyProbes reagent from Life Technologies. These cells were observed using a laser confocal scanning microscope (FV10i-LIV, Olympus, Japan).
4
Immunofluorescent Staining of Mouse Lung Sections
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Following perfusion, the superior right lung lobe from euthanized mice was excised and inflated with 4% paraformaldehyde (ThermoScientific) in PBS using a needle and syringe. Following fixation, lobes were transferred to 20% sucrose solution and then snap frozen at −80 °C in Optimal Cutting Temperature compound (VWR chemicals). Lobes were sectioned using a Cyrotome E (ThermoScientific) to 12 µm thickness and slide allowed to dry at RT overnight before storage at −80 °C. Prior to staining, slides were washed in PBS and non-specific antibody binding blocked with 3% Normal Goat Serum (Sigma) with 0.1% Triton-X 100 (Sigma) in PBS. Slides were probed with AF488-conjugated anti-mouse-CD3 at 1:100 dilution (Clone 17A2, Biolegend, cat# 100210) and AF594-conjugated anti-mouse-B200 at 1:200 dilution (Clone RA3-6B2, Biolegend, cat# 103254) to visualize T and B cells, and NucBlue fixed cell stain ReadyProbes reagent (Life Technologies) to stain nuclei. Following staining, samples were mounted with Prolong Diamond Antifade mountant (Thermo Fisher) and imaged using a BX51 microscope (Olympus, Japan) microscope.
5
Cellular Response to Amikacin-Loaded Films
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Round (22 mm) collagen-coated coverslips (BioCoat, Bedford, MA, USA) were placed into the wells of a 6-well plate for cells to adhere to. Cells were cultured at a seeding density of 0.3 × 106 to a total volume of 3 mL in each well, media included. Cells were allowed 48 h to reach confluency. Controls did not contain EPS samples. For the exposure experiments, 100 µL of crude EPS in molecular biology-grade water (5% w/v) was added to the well.
At 12 and 36 h, cells were fixed before either actin or nuclear epifluorescent stain was applied. For controls, cells were stained, but were not subject to EPS exposure. All images were not doctored in any way. All photographs of F-actin were taken using the same microscope camera settings. All photographs of DAPI staining were taken using the same microscope camera settings. Images were acquired using an Olympus IX50 inverted fluorescence microscope (Olympus, Japan). ActinRed™ 555 ReadyProbes™ reagent, NucBlue™ Fixed Cell Stain ReadyProbes™ reagent, and Image-it™ kit (Life Technologies Corp., Eugene, OR, USA) were used to examine the cellular structure of the keratinocytes and fibroblasts when exposed to the amikacin-loaded film.
At 12 and 36 h, cells were fixed before either actin or nuclear epifluorescent stain was applied. For controls, cells were stained, but were not subject to EPS exposure. All images were not doctored in any way. All photographs of F-actin were taken using the same microscope camera settings. All photographs of DAPI staining were taken using the same microscope camera settings. Images were acquired using an Olympus IX50 inverted fluorescence microscope (Olympus, Japan). ActinRed™ 555 ReadyProbes™ reagent, NucBlue™ Fixed Cell Stain ReadyProbes™ reagent, and Image-it™ kit (Life Technologies Corp., Eugene, OR, USA) were used to examine the cellular structure of the keratinocytes and fibroblasts when exposed to the amikacin-loaded film.
6
Visualizing GPCMV Infection by DiO Labeling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
DiO (Vybrant cell-labeling solution, Molecular Probe) was added to a GPCMV WT stock (4.1 × 107 PFUs in 180 μl of PBS) that was partially purified by sucrose gradient centrifugation15 (link), at a final concentration 2 μM, and the mixture was incubated for 1 hr at room temperature. The mixture was used as the DiO-labeled GPCMV stock. GPE-7 cells in 8-well chamber slides (Nunc) were incubated with the DiO-labeled GPCMV at an MOI of 22.5 for 1 hr at 4 °C, and then the inoculum was replaced with warmed culture medium. The cells were incubated for the indicated duration at 37 °C, fixed with 3.7% formalin, stained with DAPI (NucBlue Fixed Cell Stain Ready Probes reagent, Life Technologies) for detection of nuclei and with 300-fold diluted acti-stain 555 fluorescent phalloidin (Cytoskeleton, Inc.) for detection of the polymerized form of F-actin, and observed under a confocal microscope. Images of 10 fields (0.17μm2/field) for each set of conditions were captured, and the virus signals were counted.
7
Visualizing Adipocyte Differentiation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Preadipocytes were seeded and differentiated on PerkinElmer 96well black clear bottom plate. On the last day of the differentiation cells were fixed with 4% formaldehyde for 20 min at 37 °C then rinsed two times with 1× PBS. Following the fixation cells were stained with Nile Red dye using a working concentration of 10 µg/ml and diluted in 1× PBS for 20 min at room temperature (diluted from 10 mg/ml stock solution). The dye charges the lipid droplets enabling the visualization of the pattern and size of lipid droplets in adipocytes. Cells were washed once with PBS and once with water. The nuclei were stained with DAPI special formation (NucBlue® Fixed cell Stain ReadyProbes™ reagent, Life Technologies, Carlesbad, CA, USA) and rinsed in 1× PBS again. Images were acquired with an Opera Phenix™ High-Content Screening System (PerkinElmer, Waltham, MA, USA) 40× water immersion objective at 25 °C. 488 nm laser was used for the detection of Nile Red and 405 nm laser for DAPI. Images were processed using Harmony software and analyzed by Image J software [46] . We calculated the ratio of differentiation by counting the cells containing at least two large lipid droplets (differentiated) and those that did not (undifferentiated).
8
Apoptosis and Necrosis Monitoring in CE-OOC
9
Isolation and Staining of Tumor Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A tissue cut of up to 10 mg from tumour samples was subjected to gentle pipetation with 1 ml pipete, in order to separate the cells. Tumour and CSF samples were centrifuged at 600 × g for 7 min. The supernatant fluid was gently removed, and 500 μl of PBS solution were added to the sediment cells. These steps were then repeated. After the second centrifuge, the supernatant fluid was gently removed, and 10 μl of a solution containing methanol and acetic acid (3:2) were added to the cells, followed by rapid transfer of 20 μl of the cells to the center of a slide. After 7 min, the slide was transferred through an ethanol gradient (70%, 85%, 100% for 2 min each). Two drops of NucBlue® Fixed Cell Stain ReadyProbes® Reagent (Thermo Fisher Scientific, Waltham, MA, catalog no. R37606) were diluted in 1 ml phosphate-buffered saline, and 200 μl of this 4′,6-diamidino-2-phenylindole (DAPI) solution were added to the slide. After 5 min, unincorporated DAPI solution was removed, and 10 μl of ProLong® Gold Antifade Mountant Reagent (Thermo Fisher Scientific, catalog no. P36930) were added.
10
Quantifying Melanocytes in Skin Biopsies
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immuno‐histological assessment of CDKN2A and Pmel17 were performed using an anti‐CDKN2A/p16INK4a (Abcam, ab189034) or an anti‐Pmel17 (Abcam, ab63297) antibody and DAPI counterstained using NucBlue fixed cell stain Ready Probes reagent (ThermoFisher). For comparison, fluorescent images of spot and non‐spot biopsies were captured with a Zeiss Observer.Z1 microscope (Carl Zeiss Microimaging, Germany) at equal gamma values, pixel range and exposure.
Identification/quantification of melanocytes in non‐spot and spot biopsies (N = 4 subjects for each spot type) was accomplished through MITF staining utilising a MITF antibody (C5/D5 monoclonal, Sigma 284M‐97) and a Histostain plus kit (ThermoFisher) with DAB as a chromogen according to manufacture recommendations. MITF positive cell nuclei in basal‐epidermis were counted across the entire length of the DEJ of each biopsy. To account for differences in DEJ undulation between spot types and non‐spot tissue, all counts were normalized to 2 mm of total DEJ length.
Identification/quantification of melanocytes in non‐spot and spot biopsies (N = 4 subjects for each spot type) was accomplished through MITF staining utilising a MITF antibody (C5/D5 monoclonal, Sigma 284M‐97) and a Histostain plus kit (ThermoFisher) with DAB as a chromogen according to manufacture recommendations. MITF positive cell nuclei in basal‐epidermis were counted across the entire length of the DEJ of each biopsy. To account for differences in DEJ undulation between spot types and non‐spot tissue, all counts were normalized to 2 mm of total DEJ length.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!