Question

You want to analyze the potential effects of Cisplatin, a well known chemotherapeutic agent on E-cadherin expression in ASPC1 cells at different concentrations (1, 3 and 10 µM). (15 pts)

a. Name and explain at least 3 techniques you can use for this study in detail.

b. What will be your test conditions for this study?

c. With the below information already given to you, how many microliters of the drug will you add to each condition if you are using a 10 cm dish for cell culture?

- Main stock of Cisplatin is 10 mM

- Test concentrations will be 1, 3 and 10 µM.

Answer 1

You want to analyze the potential effects of Cisplatin, a well known chemotherapeutic agent on E-cadherin expression in ASPC1 cells at different concentrations (1, 3 and 10 µM). (15 pts)

a. Name and explain at least 3 techniques you can use for this study in detail.

1) measurement of cell viability with flow cytometry

2) measurement of cell viability with membrane integrity

3) measurement of cell viability with colorimetric assay

b. What will be your test conditions for this study?

One control with out the agent, experimental treatments with the three concentrations (1, 3, and 10 uM)

c. With the below information already given to you, how many microliters of the drug will you add to each condition if you are using a 10 cm dish for cell culture?

- Main stock of Cisplatin is 10 mM

- Test concentrations will be 1, 3 and 10 µM.

Initial concentration = 10 mM

final concentration = 0.001 , 0.003 or 0.01 mM

final volume = 10 cm = 392.7 cm2 of area = 39.27 ml

volume needed = Cf * Vf / Ci

For 1 uM

volume needed = 0.001 mM * 39.27 ml / 10 mM

volume needed = 0.0039 ml = 3.9 ul

For 3 uM

volume needed = 0.003 mM * 39.27 ml / 10 mM

volume needed = 0.011 ml = 11 ul

For 10 uM

volume needed = 0.01 mM * 39.27 ml / 10 mM

volume needed = 0.0392 ml = 39.2 ul