3. While preparing for plating cells for an experiment, you counted the cells in
ID: 194208 • Letter: 3
Question
3. While preparing for plating cells for an experiment, you counted the cells in order to calculate how many to use for each culture flask. After loading the hemocytometer and microscopically observing the cells, you determine that the density is too high for a count. a. What must be done to obtain a good counting range? what volume of PBS buffer or medium must be added to 10011 of the original suspension to obtain a 1:10 dilution? b. If a sample of this 1:10 dilution is added to an equal volume of trypan blue, what is the final dilution factor? c. 4. List at least three immunological assays that can be done using cultured cells.Explanation / Answer
a. What must be done to obtain a good counting range?
If a sample of cells has a high density (a lot of cells per unit of volume) it will be hard to count them in the hemocytometer, therefore the solution of cells must be diluted using PBS or medium to the point the cells do not overlap and or there are too many to count them without making mistakes.
b. What volume of PBS buffer or medium must be added to 100 microliters of the original suspension to obtain a 1:10 dilution?
To obtain a 1:10 dilution you must add 1 part of the suspension to 9 parts of the solvent (in this case PBS or medium) for a total of 10 parts, therefore, you just need to multiply by 9 the initial volume to know how much solvent you must add: 9 times 100 microliters = 900 microliters. Therefore 900 microliters of PBS or medium need to be added to 100 microliters of the original cell suspension. Initial volume is 100ul x 10 (dilution factor) = 1,000 ul (final volume).
c. If a sample of this 1:10 dilution is added to an equal volume of trypan blue, what is the final dilution factor?
1:20. Explanation: The final volume of the 1:10 diluted cell suspension is 1,000 microliters (1 milliliter), adding an equal volume will result in a final volume of 2,000 microliters (2 milliliters), and taking into account that the volume of the original suspension is still 100 microliters, 100 ul (initial volume) x 20 (dilution factor) = 2,000 (final volume).
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