If we have a plate of stem cells consist of one million cells in the start of in vitro differentiation process, after the induction of differentiation factors, and after the end of differentiation,
How many cells usually remain? (one million?)
What happens for cell cycle and cell division process? These process will be stop during differentiation?
Is there any publication about the genetics ans cellular characteristics of the cells after in vitro differentiation?
There are lot papers which described about cell cycle activity during differentiation. You are right that cell proliferation need to be stop, so cell can go for differentiation.
To track the cell proliferation, you can use FLOW CYTOMETRY analysis for Ki67, which is prominent proliferation marker, also PCNA & MCM2 expression. Even several dyes are available such as BrdU, EdU can be used.
Ki67 expression before and after differentiation will give very good answer whether cell exit the cell cycle or not...
I agree with Saurabh and Wolfgang.
Aside flow cytometry, You may also want to try the high content analysis and imaging microscopy to ascertain and measure newly synthesize DNA.
Fluorescent tracking dyes (either protein or membrane dyes) can also be used to monitor the proliferation such as celltrace violet.
Some kits are also available for checking cell proliferation. You check Thermo Fischer Scientific for such kits.
bromodeoxyuridine incorporation then use the Developmental Studies Hybridoma Bank Iowa University G3G4 monoclonal antibody against bromodeoxyuridine (it only cost $25 and works beautifully, if you can stretch to spending a few dollars more buy the concentrate). Cheap and easy way to identify and localise dividing cells using histochemical or immunofluorescent staining.
A good way of determining cell proliferation is to add CFSE into cell culture and measure the cell proliferation on the cells that you have marked with any antibody. CFSE dilutes with each mitosis, and do not interfere with the organelles in the cells, so one is able to measure how many times a specific cell has divided. Such as CD33+CD34+ cells and so..
You may also use Bromodexyuridine assay, but it is a bit more complicated in comparison to CFSE Proliferation assay. Once you have optimized the amount of CFSE to use, you will be able to use it with any cell marker.
Mitotic cells go into cell rounding, while differentiating cells do not. Therefore just count and monitor the number of rounded cells periodically. The rounded mitotic cells also take up vital dyes, like Neutral Red, preferentially. So you see the progress just by counting the number of red stained round cells.
I agree with previous posts. For cells proliferation you can use CFSE or PKH-26 dyes, EDU or BrdU kits (like https://www.thermofisher.com/order/catalog/product/C10337 for example), radioactive tritium incorporation, MTT assay, Ki-67 tracking. You always need "undifferentiated/undivided" cells control (for CFSE for example- cells grown in growth medium with low amount of serum 1-2%="undivided" cells which will give you starting "undividing" peak during flow cytometry analysis). Some cells lines have mitotic cells rounding during mitosis and shake off technique can be used to separate this cells (as mentioned above). For cell cycle analysis you need to synchronize cells by incubation them in low serum medium or using drugs as nocodazole. Depending on cells you use there will be always G0 or G1 arrested stem cell population that will not divide.
Hello
I agree with Natallia Mikhalkevich;
Chapter Monitoring Cell Proliferation by Dye Dilution: Consideration...
Article Cell Proliferation, Movement and Differentiation during Main...
Good Luck
another non toxic option would be using Alamar Blue or CCK-8 kits but you would have needed to have undertaken a standard curve assay with the cells prior to seeding.
Due to both being non toxic you could undertake multiple counts then on the same cells (wells) at different time points during the differentiation process.
- Badges
- Science topic
- Similar topics
- Linguistics
- Composition Studies
- Publication