Question

a. You start a culture of stem cells and you use a protocol to differentiate them into beta cells. To make sure they are truly beta cells.
First you want to check whether the insulin gene is expressed at higher levels in the differentiated vs. the undifferentiated cells. What methods that we studies would you use and describe the step by step procedure.

b. Then you want to demonstrate that they secrete insulin when you place glucose in their culture media. What methods that we studies would you use and describe the step by step procedure.

c. Finally, you want to show that inside differentiated cells insulin is located within secretory vesicles and quantify in which percentage of the cells. What methods that we studies would you use and describe the step by step procedure.

Answer 1

a. To check whether the insulin gene is expressed at higher levels in the differentiated vs. the undifferentiated cells, Real-time PCR is used. This technique can show the difference between the differentiated and the undifferentiated cells in the form of graph.

Steps:

i) Isolation of mRNA from both the samples.

ii) Reverse transcription to produce cDNA copies of the mRNA.

iii) Amplification of insulin coding cDNA through real-time PCR.

From the real-time PCR data, we can determine whether the insulin gene is expressed at higher levels in differentiated cells than the undifferentiated cells.

B. Immunostaining can be used to demostrate that they secrete insulin when you place glucose in their culture media. This technique is used to quantitatively analyse a sample for a particular protein, in this case it is insulin.

Steps:

i) Culture the cells in the presence of glucose.

ii) Take a small amount of the culture media without cells and place it in a sterile glass slide.

iii) Treat the slide with primary antibody that targets insulin .

iv) Wash the unbound antibody.

v) Treat with secondary antibody that is labeled and targets primary antibody.

vi) Detect the signal (colour change or fluroscence).

C. Fluorescence activated cell sorting (FACS) is used to quantify the percentage of cells containing insulin.

Steps:

i) Transfect the insulin secreting cells with fluorescence protein coding gene.

ii) Sort the cells using FACS based on the presence of the fluorescence protein.

iii) Calculate percentage of cells containing insulin by the following formula.

Percentage of cells containing insulin = (Number of cells with the fluorescence protein / Total number of cells) * 100