Dear Lisandro,

If you study a cell line and you don't know some it's characteristics , you can make a growth curve. You can seed cells diffrent number to flassk or plates. For example; you seed 50000 cells at each 6 flasks (a flask/ per day/six days) and you seed 100 000 cells at another each 6 flasks and you seed 200 000 cells at another each 6 flasks ( ıf you want, you can increase or change number of initial cells). Afterword, same time each day during six days,firstly you should take a photo a flask , then detached and count cells by trypan blue. So that, after six days, you can estimate percentage of confluency according to cell number and looking. Which number of cells which show what percentage of confluency. ( f.e; when the cells number are 500000 , confluency is %50 like that). In addition, You can calculate doubling time of cells and when your cells confluency time completely. You draw a growth curve based on data and take notes on curve. For example;

number of cells---300 000 (count by trypan blue) -------->percantage of confluency -----%30 (according to photo)

500 000 %50

1000 000 %90 or %100

If you want a number, suspend the cells per your normal lab protocol and use a hemacytometer to count them. You should be able to calculate the number of cells in the flask. Although most should be alive, I also suggest staining with trypan blue so you can exclude dead cells.

u can use cell counter

Thank you George and Firdose for your answer. Maybe I wasn`t clear with my request. I am going to do an example: X protocol say: you have to detach the cells when they are in a confluency between 50-60%, ¿How know I when the cells are in such confluency? I could be an estimation but is a little subjective, thereby I would like know if there are mathematical approaches to know the exact confluency. Other example is when you want to do a following of your culture or when you want to do a subculture. In such cases, I can`t do cell count in a hemacytometer. Let me know if I was clearer this time. Thank you very much.

I think quantitative estimation is not possible in this case. You have to harvest the cell if you want to estimate the cell number. Eye estimation may be the possible way to estimate. If you want a mathematical data, then you have to use a hemocytometer and trypan blue to count the cell and then with surface area of the flask/ plate you can make a standard curve form which confluency may be calculated.

If you want a formula to predict how many cells can give a x confluence you won't find because it varies for each different cell (primary or cell line). The best way is to try... seed a certain number of cells and observe. For example: 2x10ˆ5 cells give a 50% confluence (yes it is visual estimation and subjective), next day 75%... and then you will have an idea. If you want to be more precise count the number of cells in a hematocytomer and make a relationship x number of cells gives a y confluence.

If you are not really sure about confluence keep a track of your cells day by day until it reach 100% confluence. The surface of you flask/plate will be all covered and you'll find cells detached.

Thank you everybody for your valuable coments. I`ll keep in mind the sugestions that you are done.

Rule of thumb:By comparing the amount of space covered by cells with the unoccupied spaces you can estimate percent confluency.

I do not know of any straight forward method for measuring cell confluency other than subjectively. However, I suspect that most times the critical points to avoid are low density (there is a delay on cell duplication due likely to a lack of growth factors and cyclins concentration released to the medium) and high density (many cell types but not all, have a contact inhibition growth). That is why most protocols advice to harvest cells at 80% confluence, in other words, most likely the exponential phase of growth for most cell lines. This exponential phase is on for a longer period than just the "80%" and that is probably why the gold standard method is still the human eye. As long as you move away from low confluency (around 50% perhaps) and high confluency (>90%) you should be OK for most experiments and furthermore there should be consistency among them regardless of a hypothetical 3% confluence deviation carried from the first harvests. This is my modest opinion anyway.

Dear Lisandro

It's easy, each cell line has a definit cell density,for example BHK or Vero cells have desity of 500,000 cell per Cm2,By counting the cells according to standard cell density you can calculate percent of confluency.If you want more detail don't hesitate.

Best wishes

Alireza

Dear colleagues ,

I hope you could help me find where the error is in my failed experiment. I induced second degree burn on the skin of adult rat. I used this skin for primary culture. I repeated this trial twice and every time ,it was acomplete failure; No cells at all. Has anyone passed by a similar experience?

Dear Lisandro,

If you study a cell line and you don't know some it's characteristics , you can make a growth curve. You can seed cells diffrent number to flassk or plates. For example; you seed 50000 cells at each 6 flasks (a flask/ per day/six days) and you seed 100 000 cells at another each 6 flasks and you seed 200 000 cells at another each 6 flasks ( ıf you want, you can increase or change number of initial cells). Afterword, same time each day during six days,firstly you should take a photo a flask , then detached and count cells by trypan blue. So that, after six days, you can estimate percentage of confluency according to cell number and looking. Which number of cells which show what percentage of confluency. ( f.e; when the cells number are 500000 , confluency is %50 like that). In addition, You can calculate doubling time of cells and when your cells confluency time completely. You draw a growth curve based on data and take notes on curve. For example;

number of cells---300 000 (count by trypan blue) -------->percantage of confluency -----%30 (according to photo)

500 000 %50

1000 000 %90 or %100

I know it's an old thread, but a quick and rigorous way to do this would be to have a digital camera connected to the microscope, take several pictures and pass them trough a segmentation algorithm, that would then allow to precisely calculate the fraction of the image area covered by cells.

It might take some development, but once set it would be a quick method and certainly guarantee reproducibility. Not many inspection microscopes in culture facilities are fitted with a camera and a computer, though. And the subjective estimation method has brought us this far nicely at cost 0.

But, again, the rigorous method to determine confluency is direectly measuring the surface covered by cells.

This table might help you

You can find the answer I gave to a similar question by following https://www.researchgate.net/post/How_do_I_estimate_the_cell_confluency_percentage_in_cell_culture_flasks 

try this: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4487325/#sec0005title

You can take an image and use image J to estimate the percentage.