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Life Sciences => Cells, Microbes & Viruses => Topic started by: NoxiousPathogen on 21/02/2019 10:54:12

Title: Setting up a cell suspension, Help needed!
Post by: NoxiousPathogen on 21/02/2019 10:54:12
Currently I'm taking part in a group project, we have been set the following task for setting up a cell suspension. After being told that the cells need to be changed from 1x10 to the -5 cells per ml. We have been told this is not something that is possible via a serial dilution. Lab work is not my strongest point and could use some help understanding the following that has been provided. Many thanks for any help in advance  :)

For all stages this needs to be performed aseptically

You need to set up the 96 well plates for all experiments at 1x104 cells per well in 100 μl of phenol red free culture medium (phenol red free medium is used as this can have oestrogenic actions that interfere with the results).

Trypsinise and count your cells in the 75 cm2 that will be given to you in the practical as per last week. You now have have to adjust your cell density to 1x105 cells/ml, as you are going to add 1x104 cells per well in 100 μl of phenol red free medium. You therefore need to calculate the dilution factor that enables you to adjust your cell suspension to 1x105 cells/ml. This is achieved using the following equation

Cell density in your suspension per ml / 1x105 = dilution factor

e.g. if you had a cell suspension containing 1x106 cells/ml the calculation would be

1x106/1x105 = 10

In this case you would need to dilute your cell suspension 10-fold to get it to 1x105 cells/ml i.e. you add 9 ml of medium per ml of medium in the flask with the cells in.

You would then add 100 μl of this suspension to relevant wells in the plates trying not to create any bubbles.

Title: Re: Setting up a cell suspension, Help needed!
Post by: chris on 25/02/2019 16:08:49
So what do you want to know?

You are correct that if you have a cell suspension containing 1 x 105 cells/ml then every 1 ml of the suspension will, on average, contain 105 cells.

Therefore, if you need 104 cells in a well then you'd take 104 / 105 = 0.1 ml of the cell stock per well.
Title: Re: Setting up a cell suspension, Help needed!
Post by: Bored chemist on 25/02/2019 19:20:42
After being told that the cells need to be changed from 1x10 to the -5 cells per ml.
Srsly?