Flow Cytometry

Question 1

What are the four stages of Flow Cytometry

Answer 1

1) Cells are funnelled one at a time into the machine
2) A laser is fired at each individual cell passing through
3) Optics gather lights emitted from the cells
4) Detectors converts light into a voltage to be viewed.

Question 2

Why would a cell have greater Forward Scatter?

Answer 2

Because it is a large cell

Question 3

Why would a cell have greater Side Scatter?

Answer 3

Greater internal complexity or granularity

Question 4

Identify the following cell’s scatter profile by their name:

Neutrophils
Lymphocytes
Monocytes

Answer 4

Neutrophils

• Medium to large forward scatter
• High Side Scatter

Lymphocytes
- Small forward and low side scatter

Monocytes

• Medium forward scatter
• Low side scatter

Question 5

How does a Flow Cytometer phenotype cells?

Answer 5

• When looking to classify CD markers, the first step is use an antibody that will bind to the receptor on a specific cell that you are looking for. (for example AntiCD3 antibody).
• Fragment crystalline portion of the anti body is then binded to a fluorochrome.
• Fluorochromes fluoresce when in contact with a laser, and emit a specific wavelength that is used to signal which marker the cell is.

Question 6

Why are classical monocytes refered to as CD14-CD16++

Answer 6

They have many receptors to bind with the anti-bodies, which in turn bind with more fluorochromes, which in turn mean greater light is emitted when in contact with the laser.

Question 7

What are the four quandrants when phenotyping Lymphocytes?

Answer 7

Upper left (CD3 negative; CD4 Positive) - Monocytes

Upper Right (CD3 positive; CD4 positive)- T helper

Lower Right (CD3 positive; CD4 negative) Cytotoxic T cells

Lower left (CD3 negative; CD4 negative) - B cells or NK cells