In general from a confluent flask first I trypsinize and pellet down the cells, re suspend in 2 ml of media.Then I take 100ul of cell suspension and add 4 volume of trypan blue.After 5 mins, the cells are counted and from the average no of viable cells per square I then calculate cell density, total viable cells per sample [ viable cells per ml * 2]. So if 100% con-fluency, I get x no of cells then in 30% con-fluency, the cells no would be [ x * 30 / 100 ] . Suppose cell density is Y so Y no of cells present in 1000ul. so I can calculate [ x * 30 / 100] no of cells present in how much ul. Then I take that much ul of suspension and add rest volume of media.
But every time when I seed cells next day my observation is more no of cells in flask than my expected con-fluency. Is there any calculation error that cause this problem? How can I solve that ? Is equal no of cells or equal density of cells we should seed at the start of any experiment ?
Hi Maynak your calculation would be correct if the cells were dividing at a uniform rate.
However this is likely not to be the case. As when cells become more confluent they become more "contact inhibited".
https://en.wikipedia.org/wiki/Contact_inhibition
When cells are not touching they will grow faster than when they "begin to touch" so the growth rate is more of "sloping curve" than a linear curve (like a qPCR curve with a linear section which tapers off flat at the top).
Also to expand a little on one of Margarethe's points: when cells are at a lower density they can sometimes "stretch" out more on the dish (looking to find other cells perhaps :)). Conversely when confluency is high (and growth rate lower) they can "compact" down as the monolayer is becoming confluent.
My best advice would be to seed a number of your dishes/wells at different densities to determine the correct "confluency" for the time of your experiment with known "cells/cm2". i.e. 4000 cells per cm2, 6000 cells/cm2, 10 000 cells/cm2, 20000 cells/cm2
Using cells/cm2 also means that if you move to other formats (T25, T75 etc) this density should be transferable to those dishes (one caveat of this though is that cells/cm2 does not take into account different "edge" effects).
To improve your experiments further you might also want to stick to the same media volume: surface area ratio too. I generally use 0.3 ml of media per cm2 of the dish - some use 0.2 ml per cm2. For example if you are using 6 well plates (9.5cm2 per well) add a total volume of 2.85 ml f media (0.3*9.5).
This means that the O2/nutrient availability for the cells is more consistent between experiments.
I hope this helps,
Gary
Hi Maynak,
I assume that you desire to have cells at 30% confluency at about 12 hours after plating? I also assume the parent dish has 100% confluency at the time of trysinization, and the same size as the dish that you seed into.
Since cell confluency depends on (a) division time and (b) cell size, and both of these depend on the culturing conditions (substrate, medium, temperature), it cannot simply be calculated based on the seeding density. It is rather, a value that is determined by experience.
For this, I recommend to have a trial where you compare dishes with different seeding densities (cells/cm2 or cells/dish) and then test the next day which of these will come closest to the confluency that you desire.
Also, be aware that cell density (number of cells in a certain area) is not identical to confluency (the area that is covered by cells, independent of the cell number), therefore your calculations dont really make sense.
Thanks a lot.My formula by which numerically I calculate cell seeding density is correct or not ? How should I calculate how mwny cells should I seed for my experiment ?
Hi Maynak your calculation would be correct if the cells were dividing at a uniform rate.
However this is likely not to be the case. As when cells become more confluent they become more "contact inhibited".
https://en.wikipedia.org/wiki/Contact_inhibition
When cells are not touching they will grow faster than when they "begin to touch" so the growth rate is more of "sloping curve" than a linear curve (like a qPCR curve with a linear section which tapers off flat at the top).
Also to expand a little on one of Margarethe's points: when cells are at a lower density they can sometimes "stretch" out more on the dish (looking to find other cells perhaps :)). Conversely when confluency is high (and growth rate lower) they can "compact" down as the monolayer is becoming confluent.
My best advice would be to seed a number of your dishes/wells at different densities to determine the correct "confluency" for the time of your experiment with known "cells/cm2". i.e. 4000 cells per cm2, 6000 cells/cm2, 10 000 cells/cm2, 20000 cells/cm2
Using cells/cm2 also means that if you move to other formats (T25, T75 etc) this density should be transferable to those dishes (one caveat of this though is that cells/cm2 does not take into account different "edge" effects).
To improve your experiments further you might also want to stick to the same media volume: surface area ratio too. I generally use 0.3 ml of media per cm2 of the dish - some use 0.2 ml per cm2. For example if you are using 6 well plates (9.5cm2 per well) add a total volume of 2.85 ml f media (0.3*9.5).
This means that the O2/nutrient availability for the cells is more consistent between experiments.
I hope this helps,
Gary
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Molecular Biological Techniques