Instructions for Experiment Part 2 (EI5 Fluorescence Microscopy)

Instructions for Experiment Part 2 (EI5 Fluorescence microscopy)

These instructions describe the steps you should follow to:

i.  visualise the mitochondrial Ca2+ and NADH responses of cells caused by a specific hormone concentration

ii.  measure the integrated cellular response

iii.  enter the value you obtain in the spreadsheet.

You need to remember that the HEK cells were stimulated with four different hormone concentrations. To be able to derive a dose-response relationship, you need to quantitate cell responses caused by each of the hormone concentrations. Note: You need to analyse the same cell(s) as you did for Part 1.

Protocol

Step 1. Select a cell from the image. On the top right, you will see plots of cytosolic Ca2+, mitochondrial matrix Ca2+ and NADH recorded during the periods of hormone application (and the 60 seconds just prior to hormone application). Click on one of the mitochondrial Ca2+ plots for a particular cell. It will be displayed, enlarged, in the integrated response tool window.

Step 2. Using the integrated response tool, calculate the integrated mitochondrial Ca2+ response for the plot in the window. As before, you will need to give values for the integration period, as well as the baseline Ca2+ value.

Step 3. Enter the integrated mitochondrial Ca2+ response value in the spreadsheet.

Step 4. Repeat Steps 2 and 3 for the same cell, but at a different hormone concentration. You need to be careful to make sure you enter the data points in the correct place on the spreadsheet.

Step 5. Once you have calculated the integrated mitochondrial responses of a cell to each of the four hormone concentrations, choose a different cell and do the same again. Remember to analyse the same cell(s) as you did for Part 1. Data from 11 cells are available. You can choose how many cells you wish to analyse. In addition to calculating the integrated responses, try to consider the types of responses the cells are showing. For example, was the response transient, oscillatory or sustained? For responses that were oscillatory, was there an effect of hormone dose on the oscillation frequency?

Step 6. When you have finished analysing the cell responses, use the spreadsheet provided to calculate the mean and standard error of the mean for each hormone concentration and plot your averaged data versus hormone concentration as a graph. Use a graph with the same format as you used for plotting cytosolic Ca2+ responses.

Step 7. Repeat the analysis for NADH levels in the same cell(s) that you chose for cytosolic and mitochondrial Ca2+ responses. Calculate the mean and standard error of the mean and plot your averaged data versus hormone concentration in the same way that you have plotted cytosolic Ca2+ and mitochondrial Ca2+ responses.