Question

Given:

• 11 ml of splenocytes with concentration of 9.5 x 10^6 cells/ml
• diluting buffer= RPMI/2% FCS

What needs to be made?

• 4 plates with 96 wells each

What are the requirements?

• Each well will contain - 4 x 10^6 cells with the volume of 105 microlitres

How do you create a cell sample given the information above?

Answer 1

Given the concentration of splenocytes = 9.5 *10⁶ cells/mL

We are asked to prepare a cell concentration of 4 * 10⁶ cells in 105 uL volume.

The per mL concentration required would then be ; 1000 / 105 * 4 * 10⁶ = 9.5 * 4 * 10⁶ = 38  * 10⁶ = 3.8 * 10⁷ cells per mL

So, we need a concentration of 3.8 * 10⁷ cells per mL so that when we aliquot 105 uL we get a concentration of 4 * 10⁶ cells.

Now we need to prepare this into 4 plates with 96 wells, so 4 * 96 = 384 wells * 105 uL = 40320 uL or 40.32 mL.

We need a total of 40.32 mL, let us make it rounded to about 45 mL with a cell concentration of 3.8 * 10⁷ cells per mL so that we can aliquot 105 uL in 4 plates.

So, for a total of 42 mL, the cell concentration required would be: 42 * 3.8 * 10⁷ = 159.6 *10⁷ = 1.6 *10⁹ cells per 42 mL.

We know the original cell concentration to be: 9.5 *10⁶ cells/mL

The total volume is given as 11 mL, so the total number of cells in the original culture = 9.5 * 10⁶ * 11 = 105.6 * 10⁶ = 1.1 * 10⁸ cells / 11 mL .

This appears to be less than that calculated for the amount of cells to be transferred into 4 plates.

Let us find the amount required per plate:

96 * 105 = 10080 uL = 10.1 mL, let us round it off to 15 mL of the desired concentration.

To get 15 mL of the desired diluted culture, the number of cells needed would be : 15 * 3.8 * 10⁷ = 57 *10⁷ = 5.7 * 10⁸ cells.

We can see that even this is less than the number of cells available in the original culture!