- Q.301 – Do cells proliferate on NCP?
Most cancer cell lines proliferate at a rate equivalent to monolayer culture or slightly less. Meantime, in case of normal cells, most of which form spheroids, but does not proliferate much.
- Q.302 – How can I harvest the spheroids?
No need to use cell stripping reagents such as trypsin. As spheroids do not adhere strongly to the culture surface, it can be recovered by pipetting with medium into centrifuge tube. Precipitate the cells by centrifuging about 5 minutes at 200xg. Please avoid strong pipetting when it is needed to keep spheroid morphology.
- Q.303 – How to prepare RNA or protein from collected spheroids?
Please keep the plate on ice while collecting the spheroids. Use cold medium to recover spheroids and put into centrifuge tubes. Centrifuge at 200xg for 5 minutes at 4oC. Please extract RNA or protein from the cell pellet by using commercial kits according to your experiments.
- Q.304 – What type of cell growth assay is recommended?
The most basic and accurate way is to disperse spheroids into single cells and count the living cell number. We recommend CellTiter-Glo assay kit by Promega for the cell growth assay by measuring ATP contents.
Reference: Application notes No.11
- Q.305 – Can I use MTT or WST-8 for cell growth assay?
Those can be used by dispersing spheroids into single cells as they do not penetrate inside the spheroids
- Q.306 – Is NCP compatible with microplate reader?
NCP is compatible with SBS standard, and be used with various plate readers.
- Q.307 – Can NCP be observed with differential interference contrast microscope?
Spheroids formed on NCP can be observed by differential interference contrast microscope.
- Q.308 – How can I count the spheroid number?
By using microscope, colony counter, or high-content screening equipment, which can clearly observe and take picture of the whole well, can analyze spheroid number or size from the image.
- Q.309 – I want to test drug sensitivity screening with NCP. When is the suitable timing to add compounds or factors to the wells? Also, let me know if I need any precaution.
In case of evaluating the compounds that is effective during the period when single cells migrate and adhere into cell cluster (spheroid), add compounds at the same time when seeding. Meantime, in case of evaluating the effect after spheroid formation, add compounds 3-5 days after spheroid formation. We recommend adding small amount of high concentration solution, when adding compounds or factors after spheroid formation. As a guideline, please prepare 10 times concentration compounds, and add 1/10 volume of the culture volume. Please let the compounds disperse naturally, because pipetting after adding of compounds may cause spheroids to fall apart or to cause uneven distribution.
- Q.310 – Can NCP be used for subculture?
Yes, it is possible. Dispersing spheroids into single cells and seeding again into new well will form spheroids again. Also, it can be subcultured just by changing medium in spheroid form and not dispersing, or can also be subcultured into new well after recovering in spheroid form into centrifuge tube and changing medium.
- Q.311 – Can spheroids be immunostained?
Yes, it can be immunostained either on NCP or after moving to culture slide. In case of spheroid detachment during cell washing process, it can be stained inside the centrifuge tube. However, please be careful in case of spheroids with weak cell-to-cell adhesion, which may fall apart during cell washing process. Further, in case of spheroids with strong cell-to-cell adhesion, antibody may not penetrate into the center part of the spheroids.
Reference: Application notes No.12
- Q.312 – Can I transfect gene or siRNA?
Yes, it is possible to transfect gene or siRNA into cells before seeding or at the same time of seeding. Though we have not evaluate gene transfection after spheroid formation, it should be difficult to transfect gene into cells in the center part of the spheroids.
- Q.313 – Can I analyze cells by flow cytometry assay after dispersing?
Yes, it is possible to analyze cells with flow cytometry by labeled antibody. Please use our spheroid dispersion solution for dispersing spheroid into single cells.
- Q.314 – Can I make a slice of spheroids?
Yes, it is possible to make a slice of spheroids by freeze-sectioning using CMC or OTC compound, or a paraffin embedding method.
- Q.315 – Can I co-culture on NCP?
Yes, it is possible. You can co-culture and observe morphology at the same time, by labeling cell types into different colors with fluorescent dye.