Introductory Contents Further reading Units 	Introduction  The flow cytometer 	Data analysis 	Immunofluorescence 	Some clinical applications  DNA and the cell cycle Other applications Cell proliferation Cell death Assignment 1 Assignment 2

Assignment 2

Instructions

When you have completed this assignment, you should send the results to Michael Ormerod by email.

Your answers need only be brief; there is no need to write an essay.

The written answers should be returned as a Word file.

The layout of the data in Questions 1, 4 and 5 should be sent as a PDF file.

When you have completed the assignment satisfactorily, you will have completed the on-line component Diploma Course. Congratulations!

There are five questions

Question 1

Go here to access the data file..

The file contains data recorded for a DNA cell cycle analysis. The cells were labelled with propidium iodide so that the DNA fluoresced red.

Display a dot plot of DNA peak versus DNA area.

Draw a region (polygon) around the single cells, excluding the doublets and clumps.

Display a histogram of DNA area.

Gate the histogram on the region drawn on the dot plot.

Save the resulting layout as a pdf file, which you should submit as your answer.

Question 2

Write a simple protocol for determining the absolute number of CD4+ lymphocytes in a sample of peripheral blood. Is your protocol a single or a dual platform method? Explain the difference

Question 3

Outline an experiment to measure changes in Ca2+ concentration in CD4+ human peripheral blood lymphocytes upon the addition of an activation factor.

What dyes will you use? How will you stain the cells? Which laser(s) will you use? What parameters will you measure?

Question 4

File, Assess 3, was given to me by Robert Sutherland, Toronto. It is an apheresis sample from a patient after mobilisation for 5 d with G-CSF. The peripheral blood cells have been labelled with CD45-FITC and CD34-PE.

Go here to access the file.

On the dotplot of 7-AAD versus Forward Scatter. set a region, (live), to exclude the 7-AAD +ve cells (These are the dead cells and are the brightest  population). The live cells are negative for 7-AAD.

Display a dotplot of Side Scatter versus Forward Scatter. Set a generous region around the lymphocytes (lymphs).

Display a dotplot of CD34 versus CD45. Gate the dotplot to include cells in regions live and lymphs. Place quadrant regions on the dotplot to include the CD45+ve, CD34+ve cells in the Upper Right quadrant.

Record the number of CD34 +ve cells as a percentage of all the leucocytes in the sample (that is, all the white blood cells, not just the lymphocytes).

Save the layout as a PDF file and submit with your answer.

Question 5

File, Assess.4, comes from George Wilson, Detroit. Chinese Hamster, V79, cells have been labelled with BrdUrd for 30 min and then incubated without BrdUrd for 4 h. The cells were then fixed and labelled with anti-BrdUrd-FITC and PI.

Go here to access the file.

On a dotplot of BrdUrd-FITC against DNA area, set regions to measure:

Express the two numbers as percentages of the total number of cells in the sample. Place these percentages on the dotplot.

Save the layout as a PDF file and submit with your answer.