1. Referring to the simplified schematic of the flow cytometer below, assign the appropriate letter to each of the components:

2. What is meant by hydrodynamic focusing?

3. What is Forward Scatter (FSC) and what does it measure?

4. What is Side Scatter (SSC) and what does it measure?

5. A peripheral blood sample was subjected to flow cytometry and the following scatter-plot was obtained.

6. The sample in question 5 was treated according to the protocol below before being subjected to flow cytometry.

- Pre-warm the whole blood treatment buffer* to 37°C in a water bath.
- Add 2mL of whole blood treatment buffer to 100µL of whole blood.
- Mix by inversion or gentle vortexing.
- Incubate at room temperature for 10-15 mins.
- Centrifuge at 400g for 5 minutes
- Remove the supernatant and resuspend the pellet in Ca/Mg-free PBS.

*Whole blood treatment buffer - 155mM NH4Cl, 1mM KHCO3, 0.1mM EDTA, pH 7.2

a. What was the purpose of adding the whole blood treatment buffer? Hint - what cell type is missing from the scatter-plot in question 5?

b. Can you think of another method to achieve the same aim?

c. Bonus question (email J.Pakay directly - the first correct, detailed answer from each prac day wins a chocolate bar). What is the mechanism of action of whole blood treatment buffer?

7. What is the point of setting a threshold level for forward scatter when analysing flow cytometry data?

8. The following histograms were produced using flow cytometry after labelling B-cell lymphocytes with propidium iodide. Histogram A is from a healthy individual while Histogram B is from a patient displaying severe anaemia and enlarged lymph nodes.

a. What does propidium iodide bind to?

b. Why has a linear scale been used for the x-axis rather than a logarithmic scale?

c. Label which stage of the cell cycle each of the cell populations are in Histogram A.

d. Explain the result in Histogram B. Speculate on the possible condition of the patient.

9. HeLa cells were treated with a new experimental drug (thought to inhibit BCL-2) to rule out any potential cytotoxic effects. Cells were exposed to the drug for 24 hours prior to harvesting (B) or a vehicle control (A). The cells were then harvested and stained with Annexin V - FITC and propidium iodide (PI) before analysis by flow cytometry. Fluorescence due to FITC is measured by the FL1-H channel and fluorescence due to PI is measured by the FL3-H channel.

a. Annexin V can bind to phosphatidylserine. What does an increase in Annexin V binding represent?

b. What percentage of cells are viable in the vehicle control?

c. What effect does the drug have on HeLa cells?

d. What effect might over-expressing BCL-2 in the HeLa cells prior exposure to the drug have?