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Posted: December 17th, 2022

Biol 477/L – Cell and Tissue Culture

Biol 477/L – Cell and Tissue Culture
Lab Exam 2 (75pts) Name_______________________________
1. (15 pts.) You are given a 100mM stock solution of GC7 (an eIF5A inhibitor) and you need
1ml at a final concentration of 5 nM. You are given 10 ml of diluent (DMSO). Describe in a
bullet-point format how would you make this dilution? (Remember that pipetting less than 1 µl
at a time is not very practical – so you are not allowed to do that!).
2. (15 pts.) A good friend has given you a flask of adherent cells and being an overachiever, you
want to do an experiment as soon as possible. The cells are 60% confluent and double every 24
hours. What should you do and when can you do an experiment that requires 6 flasks that are
20% confluent? You need to keep one flask growing at 20% confluence.
3. In experiment 3, you analyzed the cell cycle profile of 4T1 cells transfected with empty vector
or ALK4-overexpressing vector by flow cytometry. In theory, this experiment could also be done
using wide field fluorescence and phase-contrast microscopy as you did in experiments 1-3.
A. (10 pts.) Why is this true?
B. (10 pts.) Describe how you would evalueate the percentage of total live cells that
contained 2n vs. 4n DNA content.
4. As we discussed in class, C3H10T1/2 cells are a multi-potent mesenchymal stem cell line. As
such, they can be differentiated into fat cells (adipocytes) and the extent of differentiation can be
determined using Oil Red O staining. I need your help in doing some calculations for an
upcoming experiment in which students in my lab will be analyzing C3H10T1/2 differentiation
into adipocytes. The students will be making their own solutions.
A) (5 pts.) They will be provided with 200 proof (100%) ethanol. How should they make
10mls of a 4% ethanol solution?
B) (5 pts.) For the Oil Red O staining procedure, the students will need to make 1 mL the
final staining solution at 52.5mg/mL per sample that is going to be stained (they will be
staining 6 samples all together). They are given 1mL of a 1g/mL stock solution that the
class GA will make. Do they have enough Oil Red O stock solution for making the final
staining solution? If so, what volume of the stock solution should they dilute with water,
and how much water should they use?
C) (10 pts.) Flow cytometry is a complementary method for analyzing adipocyte
differentiation from C3H10T1/2 mesenchymal stem cells. Based upon the theory behind
the use of flow cytometry, and the fact that Oil Red O+ cells fluoresce in the FL2 (red)
cannel, draw a scatter plot of what the SSC vs. FL2 channel would look like if
approximately 50% of the total cells were successfully differentiated into adipocytes and
stained positive for Oil Red O.
D) (5 pts.) The students will need to harvest a minimum of 6×106 differentiated C3H10T1/2 cells
for their Oil Red O and Flow Cytometry protocol (for each experimental condition). For
each condition in their experiment, how many wells of a 6-well plate will they need to use, if
each well can accommodate 2×106 cells when grown to confluency? If they are given a cell
stock of these cells at 2×105 cells/mL in 10 mL of complete media and they need to run 6
conditions, how long will they have to wait after plating the cells at 1×105 cells/well (if the
cells double every 24 hrs) before they have enough cells in each condition to analyze?

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