Question

You have recently joined a lab and will start working with J558 cells today. You are handed a T25 flask (5 mL) containing these cells, so you take an aliquot of the cells (using aseptic technique) and dilute the aliquot with Trypan Blue in a 1:1 ratio. After loading the hemocytometer and observing the cells under the microscope, you count the cells and obtain the following values:

Quadrant:	Transparent cells:	Blue cells:
Q1	52	1
Q2	60	3
Q3	58	2
Q4	51	2
Q5	57	1

You want to seed these cells into as many T75 flasks (15 mL) as possible to obtain a higher cell yield in a few days for an experiment. The seeding density for these cells is 2 x 10⁵ cells/mL.

1) How many total cells do you have in the T25 flask?

2) How many T75 flasks can you seed with the cells you have?

3) How would you resuspend your cells to seed the T75 flask(s)?

4) How many T25 flasks can you seed with the remaining cells you have?

Answer 1

The formula for calculating the number of cells/ml is = average of all the cells counted from each quadrant x 10ⁿ x dilution factor

average of live cells counted = (52+60+58+51+57)/5 = 278/5 = 55.6

average of dead cells counted = (1+3+2+2+1)/5 = 9/5 = 1.8

n = number of quadrants = 5

dilution factor = 2

Hence the number of live cells/ml = 55.6 x 10⁵ x 2 = 111.2 x 10⁵ cells/ml

Total number of live cells in 5ml = 111.2 x 10⁵ x 5 = 556 x 10⁵ cells

number of dead cells = 1.8 x 2 x 10⁵ = 3.6 x 10⁵ cells/ml

Total number of dead cells in 5ml = 3.6 x 10⁵ x 5 = 18 x 10⁵ cells.

1. Total number of cells in T25 flask = number of dead cells + number of live cells = 556 x 10⁵ + 18 x 10⁵ = 574 x10⁵ cells

2. For seeding of the cells only live cells are considered. Hence to obtain a cell count of 2 x 10⁵ cells/ml in a 15ml flask, from a stock cells of 111.2 x 10⁵ cells/ml we will be needing = 2 x 10⁵ x 1000ul / 111.2 x 10⁵ = 17.9 ul (microlitres) of volume for one T75 flask.

The number of T75 flasks that can be seeded by this cells are = volume of media in T25 flask/ volume required to seed one T75 flask = 5000ul/17.9ul = 279 T75 flasks can be seeded with a remainder of 5.9ul of stock media left.

3. To seed the T75 flask we need to first add 17.9ul from the stock cells and add 14982.1ul of media in the flask.

4. With the remainder of the 5.9ul of the media left, we can seed one T25 flask as the seeding density of the T25 flask will be 1/3rd of the T75 flask seeding density. Hence 17.9ul/3 = 5.9ul.

If you have any query kindly comment before giving thumbs up. Thank you.