NIH3T3 cells were seeded overnight in a specific 96-well plate (Cyntellect, San Diego, CA, USA) for laser-enabled analysis and processing (LEAP™) (Cyntellect, San Diego, CA, USA; located at Bindley, Purdue University) at 5 × 105 cell/mL concentration in culture media. 15 µL of MWCNTs or GFs at 1 mg/mL was added to each well to a final concentration of 50 µg/mL. Then, cells were incubated for 2 and 24 h at 37 °C under 5% CO2 atmosphere. The medium was then removed, and the wells were washed thoroughly with PBS supplemented with Mg2+. Finally, calcein-AM green dye (Life Technologies, Carlsbad, CA, USA) for cell viability, DHE dye (Life Technologies, Carlsbad, CA, USA) for ROS production, and Hoechst 33342 dye (Life Technologies, Carlsbad, CA, USA) for DNA staining were added. Cells were stained for 30 min in the dark and at room temperature before being processed using LEAP. Three replicates per CBN were tested, each group has a complete field image of the well, see Additional file 1 . The percentage of ROS production was calculated from the cells expressing DHE over the total of cells labeled with calcein.
Calcein green am dye
Manufactured by Thermo Fisher Scientific
Sourced in United States
Calcein-green AM dye is a fluorescent indicator used for the detection and quantification of cell viability. It is a membrane-permeable dye that is hydrolyzed by intracellular esterases to produce a green-fluorescent calcein molecule, which is retained within viable cells.
Automatically generated - may contain errors
Lab products found in correlation
Infinite m1000 pro, by Tecan (3 mentions)
Hank s balanced salt solution, by Thermo Fisher Scientific (2 mentions)
Hoechst 33342 dye, by Thermo Fisher Scientific (1 mentions)
Dhe dye, by Thermo Fisher Scientific (1 mentions)
Facscanto 2, by BD (1 mentions)
Hanks balanced salt solution (hbss), by Thermo Fisher Scientific (1 mentions)
5 protocols using calcein green am dye
1
Evaluating Nanoparticle Cytotoxicity and ROS
2
Cytotoxicity Assessment of CNTs and GFs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were seeded in culture media overnight on 5 cm diameter Petri dishes at a concentration of 2.5 × 106 cell/mL. After 24 h, 200 µL of MWCNTs or GFs at 1 mg/mL were added to each well to a final concentration of 10 and 30 µg/mL. Then, cells were incubated for an additional 24 h at 37 °C in an atmosphere with 5% CO2 and a relative humidity of 95%. The culture medium was then removed, and the wells were washed thoroughly with PBS supplemented with Mg2+. Later, cells were harvested using trypsin and centrifuged at 200×g. Subsequently, calcein-AM green dye (Life Technologies, Carlsbad, CA, USA) and propidium iodine (PI) (Life Technologies, Carlsbad, CA, USA) were added to stain the cells for 30 min in the dark at room temperature. Finally, the stained cells were measured using a flow cytometer (FACS Canto II, BD Bioscience, San Jose, CA, USA; located at Universidad de Los Andes), which counted to 10,000 events for 30 s. Each group was seeded and tested three times, see Additional file 1 .
3
CRISPR-mediated Invasion Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
22RV1 CRISPR clones were grown in 10% CSS-supplemented medium for 48 hour for androgen starvation. Special matrigel-coated invasion chamber were used that were additionally coated with a light-tight polyethylene terephthalate membrane to allow for fluorescent quantification of the invaded cells (Biocoat: 24-well format, #354166). 50,000 starved cells were resuspended in serum-free medium and were added to each invasion chamber. 20% FBS-supplemented medium was added to the bottom wells to serve as a chemoattractant. After 12 hours, medium from the bottom well was aspirated and replaced with 2ug/ml Calcein-green AM dye (ThermoFisher Scientific; C3100MP) in 1X Hank’s Balanced Salt solution (Gibco) and incubated for 30 minutes at 37C. Invasion chambers were then placed in a fluorescent plate reader (Tecan-Infinite M1000 PRO) and fluorescent signal from the invaded cells at the bottom was averaged across 16 distinct regions/chamber to determine the extent of invasion.
4
Matrigel-Based Invasion Assay for LNCaP Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
LNCaP CRISPR clones were grown in 10% CSS-supplemented medium for 48 h for androgen starvation. A Matrigel-coated invasion chamber was used, which was additionally coated with a light-tight polyethylene terephthalate membrane to allow for fluorescent quantification of the invaded cells (Biocoat: 24-well format, no. 354166). Fifty thousand starved cells were resuspended in serum-free medium and were added to each invasion chamber. Twenty percent FBS-supplemented medium was added to the bottom wells to serve as a chemoattractant. After 12 h, medium from the bottom well was aspirated and replaced with 2 μg/ml Calcein-green AM dye (Thermo Fisher Scientific; C3100MP) in 1× HBSS (Gibco) and incubated for 30 min at 37 °C. Invasion chambers were then placed in a fluorescent plate reader (Tecan-Infinite M1000 PRO), and fluorescent signals from the invaded cells at the bottom were averaged across 16 distinct regions per chamber to determine the extent of invasion. For rescue experiments, stable lines overexpressing the NSD2 isoforms were generated. Briefly, to LNCaP NSD2 KO lines, GFP or NSD2-Long isoform containing viruses were added. These lines were then used to perform the invasion assay as described above.
5
CRISPR-mediated Invasion Assay
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!