Measuring Cell Concentrations

Question (Concentrations: cells): You want to split cells into a new flask so that the flask with have a 50 ml volume and final cell concentration of 4 * 105 . Your flask with cells has a volume of 50ml and a concentration of 25 * 104. What the volume that should be take out of this flask to end up with your desired final concentration?

(CI VI = C2 V2)     (25 * 104) VI = (50 ml) (4 * 105)

VI = 80 ml                    This volume is clearly more than what is in the flask. So we will instead have to spin the cells down and add media instead. What then should be the volume of the second flask to get the desired final concentration?

(CI VI = C2 V2)     (25 * 104) 50 ml= (V2) (4 * 105)

V2 = 31.25 ml      So spin down the flask having 50 ml and then resuspend the cells in about 32 ml.

Question (Concentrations: cells): You want 15 ml of cells at a concentration of 50,000 cells/50 ul. After doing a cell viability count, you figure that you have 280K cells/ml in a flask which has the cells suspended in about 75 ml. What volume would you need to take out of that flask to get your desired concentration?

(CI VI = C2 V2)     (280Kcells/ml) VI = (15 ml) (50 K cells/0.05 ml)

VI = 53.5 ml

Question (Concentrations: cells): After counting cells, you find that you should have 10 million in the pellet that you spun down (in this example, you came up with the # by mutiplying #cells X 2 (dilution factor) X 104 (volume of hemacytometer) X #ml (cell suspension). You want a concentration of 1 X 106. What volume should you resuspend your cells?

10ml

Question (Concentrations: cells): You have a [cells] or 1 X 106 and you would like a final [cell] of 2 X 105. What volume should you take from the 1 X 106 and place into your plate well for a final volume of 1 ml.

CI VI = C2 V2)     (1 x 106 cells/ml VI ) = (1 ml) (2 x 105)

V1= 0.2 ml or 200 ul    You should thus add 200 ul (this amount can never change!) to your 1 ml volume. So you might add 200 ul of cells + 800 ul media (this amount can change to make room for other chemicals).

Question (Concentrations: cells): You have a [cells] or 8 X 106 cells/ml and you would like a final [cell] of 1 X 106. From outlining your experiment, you determine that you need to use 13 wells in a 24 well plate (to do everything you want to accomplish). Is it possible to conduct your experiment at this concentration?

Yes. But you can not use 1 ml of you cells (brought up in 8 ml) because you will not have enough cells to distribute this way. What you must use is a smaller volume. If you use 0.5 ml instead of 1ml per well you can still keep a [final] of 1 X 106 . This would in fact allow you to use up to 16 wells. 

So now what volume should you take out of your 1 ml of cells to get this correct concentration?

CI VI = C2 V2 (1 x 106 cells/ml VI ) = (1 x 106)(0.5 ml)

V1= 62.5 ul (this can not change!)  (Add this to your LPS + MDP up to a total volume of 0.5 ml)

Question (Concentrations: dendritic cells & LP infection): You determine that you have 16 million dendritic cells/ml and after your OD reading that you have 2.9 X 109 bacterial cells/ml. You would like to infect your dendritic cells with the bacteria at a ratio of 10:1. What volume of LP should we take to get a 10:1 infection?

One way to do this is to multiply the dendritic cells by a factor of 10 and then divide by the # bacterial cells. So

16 X 107 dendritic cells/ ml / 2.9 X 109 bacterial cells/ml 

= 5.5 x 10-2 ml  or 0.055ml or 55 ml

Question (Concentration in multi-well plates): After counting cells, you find that you have 8 million cells suspended in 5 ml. You want a concentration of 1 million per 200 ul. What should you do?

CI VI = C2 V2 (8 x 106 cells/ml VI ) = (1 x 106)(0.2 ml)

V1= 125 ul 

Question (Concentration in multi-well plates): Suppose in question above that you want to set up a dilution of the cells much like an ELISA. you want to start your highest concentration of cells at 4 million.

4 million is 4x 25  so you would add 100 ul of your stock    to 100 ul media

2 million, add 50 ul        to 150 ul media

1 million, add your 25 ul    to 175 ul media

.5 million 12.5 ul    to 187.5 ul media

.25 million 6.25 ul    to 193.75 ul media

.125 million   3.125 ul to 196.875 ul media