2. A graduate student is working with CHO cells to express a kinase. He needs to
ID: 149498 • Letter: 2
Question
2. A graduate student is working with CHO cells to express a kinase. He needs to subculture the cells and start a new plate, for what he trypsinized a confluent plate of cells. The cells were resuspended in 5ml. He made a dilution of the original cell suspension with trypan blue to check cell viability and to count them. He used a hemocytometer and he counted 120 cells presen in 16 small squares of the hemocytometer. a. What is the concentration of the initial cell suspension? b. What is the total number of cells in the 5ml of the initial cell suspension? c. What volume of the original cell suspension, the student will need to seed in order to start a new culture with 1,200,000 (6X106 cells) cells? How much medium does he need to add if the final volume of the flask is 10ml?Explanation / Answer
a. 1 square has a volume of 0.1mm3
Concentration of cells after dilution
= 120 cells/16 squares
=120/1.6 mm3
= 75 cells/mm3
Since, the solution was diluted to half its original, we have,
the original concentration = 75/(1/2)
= 150 cells/mm3
Therefore the concentration of initial cell suspension
= 150 cells/mm3.
= 150,000 cells/cm3
or 150,000 cells/ml
b. Total number of cells in 5ml of solution
= 150,000*5
= 750,000 cells
c. Total number of cells required= 1,200,000
Volume of original suspension required
= (1,200,000/750,000)*5
= 8 ml
Amount of medium to be added
= 10-8 ml
= 2ml
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