In order to perform a fluorescence experiment you need to se

In order to perform a fluorescence experiment, you need to seed a 4-well chamber slide with 60,000 cells in each well. To prepare for the cell count, you transferred 20 uL of cell suspension into 30 uL of trypan blue. You calculated 19, 12, 25, 29 and 11 cells in 5 large boxes using the Neubauer hemacytometer. Based on this information, what is the cell density (cells per mL) of your cell suspension? How much cell suspension will you need to transfer roughly 60,000 cells into to each well?

Solution

In order to solve the problem, we have to calculate the following parameters -

1. Number of cells in suspension based on neubauer count
2. Accounting for dilution factor and revising the number of cells in suspension
3. Calculating the volume of suspension to be seeded for fluorescence experiment.

Part 1:

Number of cells in box 1 = 19
Number of cells in box 2 = 12
Number of cells in box 3 = 25
Number of cells in box 4 = 29
Number of cells in box 5 = 11

Average number of cells in one box = ( 19+12+25+29+11) / 5 = 19.2 = Rounded off to 19 cells

Therefore, in one box, there are 19 cells.

Now,
Volume of one box is 0.1uL. That means, 19 cells are present per 0.1uL of suspension,
Therefore, number of cells present in 1mL (1000uL) of volume = 19 * 10⁴ cells

Part 2
Amount of cell suspension used = 20uL
Amount of Trypan blue used = 30uL
Total volume = 20 + 30 = 50uL

Dilution factor = ( Total volume ) / ( Volume of cell suspension ) = (50) / (20) = 2.5
Therefore, the dilution factor is 2.5

We now have to multiply the answer which we obtained in part 1 with the dilution factor to obatin the actual number of cells present in 1 mL of suspension.

Therefore,

Actual no. of cells/mL = 19 * 10⁴ * 2.5 = 47.5 * 10⁴ cells/mL

Part 3:

Number of cells required in each well = 6 * 10⁴ cells/mL
Given concentration of cells = 47.5 * 10⁴ cells/mL
Therefore, volume of parent solution that would contain 6 * 10⁴ cells = (6 * 10⁴) / (47.5 * 104) = 0.126mL

Final Solution
In order to seed 60,000 cells per well, we need to dispense 0.126mL of parent solution (undiluted) in each well.