Confluency is not the same as cell number, it is rather the percentage of the area covered by adherent cells. HUVEC cells have a diameter of about 12 um while HeLa cells are closer to 20 um. This means that a fully covered (i.e. confluent) cell culture flask/dish of HUVEC cells will have almost twice the number of cells as a fully confluent flask of HeLa cells. From what I understand from your question, you are not really interested in the actual cell number at this point, you just want to know how to estimate the confluency.
- 50%: An easy estimation is for 50 % confluent cells since the area covered by the cells looks similar to the area that is not occupied by the cells.
- 100%: Another easy estimation is for cells that are 100% confluent since you will not see any space in between the cells. Here there are two scenarios, normal cells have contact inhibition and will rarely reach a real 100% and there are immortalized cells that do not have contact inhibition in which case they will squeeze each other and get to half their "normal" size and then start detaching from the plate and dying off (death phase). Either way a plateau will be reached before the death phase (the plateau is also called the stationary phase).
- 70% - 80% (this is what people are usually interested in): Subculturing/passaging right before the plateau, when the cells are still growing exponentially (near the end of the log phase) will result in improved overall cell viability, will yield cells that are less aggregated and will shorten the lag time (idle time before the cells start logarithmic growth once again). It doesn't have to be exact. This is an estimation and a range. You want them somewhere between 50 % and 100%, that should be easy and will become easier to estimate the more you culture cells. If you are in doubt you are most likely between 60% and 90% and the simplest thing you can do is go ahead and subculture your cells.
I hope this helps!
Hi Pegah Ardestani,
Hope you will get detailed info by looking at this link
https://www.researchgate.net/post/How_to_estimate_the_cell_confluency_in_culture_flask_plates
Hi, appropriately confluency can be estimated by observing and comparing the space occupied by the cells in the plate/flask with that of the unoccupied space under microscope. On the either side, Cell counting using tryphan blue can give you the accurate no. of cells in the plate/flask and then Compare no. of cells with the size of the plate/flask. Good Luck
Confluency is not the same as cell number, it is rather the percentage of the area covered by adherent cells. HUVEC cells have a diameter of about 12 um while HeLa cells are closer to 20 um. This means that a fully covered (i.e. confluent) cell culture flask/dish of HUVEC cells will have almost twice the number of cells as a fully confluent flask of HeLa cells. From what I understand from your question, you are not really interested in the actual cell number at this point, you just want to know how to estimate the confluency.
- 50%: An easy estimation is for 50 % confluent cells since the area covered by the cells looks similar to the area that is not occupied by the cells.
- 100%: Another easy estimation is for cells that are 100% confluent since you will not see any space in between the cells. Here there are two scenarios, normal cells have contact inhibition and will rarely reach a real 100% and there are immortalized cells that do not have contact inhibition in which case they will squeeze each other and get to half their "normal" size and then start detaching from the plate and dying off (death phase). Either way a plateau will be reached before the death phase (the plateau is also called the stationary phase).
- 70% - 80% (this is what people are usually interested in): Subculturing/passaging right before the plateau, when the cells are still growing exponentially (near the end of the log phase) will result in improved overall cell viability, will yield cells that are less aggregated and will shorten the lag time (idle time before the cells start logarithmic growth once again). It doesn't have to be exact. This is an estimation and a range. You want them somewhere between 50 % and 100%, that should be easy and will become easier to estimate the more you culture cells. If you are in doubt you are most likely between 60% and 90% and the simplest thing you can do is go ahead and subculture your cells.
I hope this helps!
@Barae Jomaa Thanks for your detailed answer.
Actually, I am working on the effect of a protein on cell proliferation, and I have to compare cell confluency among my plates during the study. This amount could be varied from 20% to 70% during the study. Do you have any Idea about how to differ the percentages which are less than 50%?
Snap a picture under the microscope and analyze it in ImageJ (freeware). Here's the download link: http://imagej.nih.gov/ij/download.html and here's an article that describes the method: http://www.sciencedirect.com/science/article/pii/S2215016114000260
Hello Pegah,
It is easy to quantitatively estimate cell confluence in phase contrast images of cells by first segmenting the cell surface fraction in Ilastik (because we figured out simple filtering doesn't work well enough) and then directly measuring confluence in imageJ (both programmes are freeware and easy to use). We have described this simple approach at: https://www.academia.edu/40969549/
@ Yordan Yordanov:
Is the pdf available on any other platform? I don't want to share my e-mail contacts to get to it ;)
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