Question

Use the information below to answer the following questions.

After taking an aliquot of J558 suspension cells from your T75 culture flask, you dilute this aliquot in Trypan Blue in a 1:1 ratio. After loading the cells on the hemocytometer you obtain the following counts:

	Transparent cells	Blue cells
Quadrant 1	32	1
Quadrant 2	31	1
Quadrant 3	30	1
Quadrant 4	31	2
Quadrant 5	33	1

1) What is the cell viability?

2) What is the density of the cells (living) in the culture after considering this new dilution with Trypan Blue?

3) How many total cells are in the T75 flask (15 mL)?

4) After spinning down your cells in the centrifuge and removing the media, how many mL of media would you use to resuspend your pellet to obtain a resuspension cell density of the total number of cells needed to seed one T25 flask per mL? How would you then seed each T75 flask?

Resuspension density = (total number of cells needed to seed one T75 flask)/(1 mL)

a) mL for resuspension (to two decimal places)=   mL
b) How many mL of resuspended cells would each T75 flask receive?  mL
c) How many mL of media would each T75 flask receive?  mL

5) First, consider how many suspension cells you would need to seed in one T75 flask (15 mL) at the seeding density for J558 cells (2 x 10⁵ cells/mL).How many T75 flasks could you seed with the cells that you have?

Answer 1

1. Cell viabily is the % of live cells, which is calculated as follows-

%viable cells= [{1-(total number of blue cells / number of total cells)}100] (eqn no.1)

Here, total number of blue cells = sum of blue cells in all quadrants= 1+1+1+2+1= 6

Here, number of total cells = sum of blue cells in all quadrants + sum of transparent cells in all quadrants=6+ 157 = 163.

Now, lets plug all these values in eqn no.1

%viable cells= [{1-(6 / 163)}100] = [{1-0.0368}100] = [0.9632100]= 96.32

So, cell viability or %viable cells is 96.32%

2. Density of viable cells is calculated as follows-

viable cells (live cells/mL)= (number of live cells counted/ number of large squares counted) dilution factor 10^4 (eqn no.2)

Here, total no. of live cells = sum of transparent cells in all quadrants= (32+31+30+31+33)=157

Here, number of squares counted =5

Here, the cells are aliquoted in trypan blue in a 1:1 ratio, this means dilution ratio is 1:2. So,dilution factor=2.

Now, lets plug all these values in eqn no.2

viable cells (live cells/mL)= [(157/5)2 10^4 ] = [31.42 10^4 ]= [62.8 10^4 ]

So, density of live cells = 62.8 10^4 cells/mL.

3. Total number of cells in the T75 flask= number of live cells/mL volume of media (eqn no. 3)

The volume of media = 15mL (given)

live cells/mL= 62.8 10^4 cells/mL (as calculated in 2nd part of the question)

Now, lets plug all these values in eqn no.3

Total number of cells in the T75 flask= 62.8 10^4 cells/mL 15mL= 94210^4 cells/mL= 9.4210^6 cells/mL.

So, total number of cells in the T75 flask is 9.4210^6 cells/mL.

5.Number of suspension cells needed to seed in one T75 flask (15 mL) at the seeding density for J558 cells (2 x 10⁵ cells/mL)= volume seeding density = 15 2 x 10⁵ cells/mL= 3010⁵ cells/mL= 3x 10⁶ cells/mL.- eqn no.4

No. of T75 flasks that can be seeded with the cells that I have= Total no. of cells I have Number of suspension cells needed to seed in one T75 flask

Here, Number of suspension cells needed to seed in one T75 flask = 3x 10⁶ cells. (from above)

Here, No. of cells that I have is= 9.4210^6 cells/mL (from 3rd part of the question)

Now, lets plug all these values in eqn no.4

No. of T75 flasks that can be seeded with the cells that I have=  9.4210^6 cells/mL 3x 10⁶ cells = 3.14

This, means 3 T75 flasks flasks can be seeded with the no. of cells that I have.