Let's break down each part of the problem step by step.

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A). How many cells total do you need in order to seed the entire plate, and in what total volume of medium will you need to dilute them (for 110 wells, including extra for pipetting error)?

1. Cells per well: You need 5500 cells per well.
2. Total number of wells: The question asks to prepare for 110 wells (96 wells + 14 extra for pipetting error).

The total number of cells you need for 110 wells is:

$\text{Total cells} = 5500 \, \text{cells/well} \times 110 \, \text{wells} = 605,000 \, \text{cells}.$

3. Final volume per well: Each well will have a total volume of 100 μl.
4. Total volume of medium: For 110 wells, the total volume of medium needed is:

$\text{Total volume} = 100 \, \mu l \times 110 \, \text{wells} = 11,000 \, \mu l = 11 \, \text{ml}.$

So, you need 605,000 cells in a total volume of 11 ml of medium.

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B). After trypsinizing cells, how much of your trypsinized stock do you need to use to plate the above experiment, and how much culture medium?

1. Cell count after trypsinization: You counted a 1:8 dilution of the trypsinized stock and found 17 cells per quadrant in a hemocytometer. Since each quadrant in a hemocytometer represents a volume of 0.1 μl, the number of cells per μl in the diluted sample is:

$\text{Cells per} \, \mu l \, \text{in dilution} = 17 \, \text{cells} \times 10 = 170 \, \text{cells/μl}.$

Since this is a 1:8 dilution, the original stock concentration is:

$\text{Cells per} \, \mu l \, \text{in trypsinized stock} = 170 \, \text{cells/μl} \times 8 = 1360 \, \text{cells/μl}.$

2. Amount of trypsinized stock needed: You need 605,000 cells for the entire experiment, and your stock contains 1360 cells per μl. The volume of stock required is:

$\text{Volume of trypsinized stock} = \frac{605,000 \, \text{cells}}{1360 \, \text{cells/μl}} \approx 444.85 \, \mu l.$

3. Volume of culture medium: The total volume for the experiment is 11 ml (or 11,000 μl). Thus, the amount of medium to add is:

$\text{Culture medium volume} = 11,000 \, \mu l - 444.85 \, \mu l = 10,555.15 \, \mu l.$

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Final Summary:

• Total cells required: 605,000 cells.
• Total volume of medium: 11 ml.
• Volume of trypsinized stock needed: 444.85 μl.
• Volume of culture medium needed: 10,555.15 μl.

Would you like more details, or have any questions?

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Related Questions:

1. How can you adjust the cell seeding if you have a lower cell concentration in your stock?
2. What steps would you take to improve accuracy in cell counting with the hemocytometer?
3. How does pipetting error impact the results of a cell seeding experiment?
4. Why is it important to account for extra wells when preparing cell suspensions?
5. How can you calculate the appropriate dilution factor if your trypsinized stock has a different concentration?

Tip:

Always mix your cell suspension thoroughly before seeding to ensure even distribution across wells.