Flow Cytommetry Lab

Question 1

Why do we need to use controls in Flow Cytommetry?

Answer 1

• They are important for REPRODUCIBLE collection and ACCURATE data analysis

1. Unstained WBC allow detection of autoflourescence
2. Isotope control antibody (for each test antibody CD19 PE or CD45 FITC) allow us to determine the NON SPECIFIC flourescence that may be caused by non specific Ab isotype binding to some immune cells
3. When performing multicolour flow cytommetry, single colour control samples are vital to determine levels of compensation

Question 2

Why are unstained WBC’s used (as a control in flow cytometry)?

Answer 2

• to determine the background flourescence (AKA autoflourescence)
• Autoflouresence is the natural emission of light by cell proteins when they absorb laser light. This control is used to tell the difference between autoflourescent portein light from light belonging from flourophore conjugated antibodies

Question 3

What is Autoflourescence? Which control is used to determine this?

Answer 3

Autoflouresence is the natural emission of light by cell proteins when they absorb laser light. This control is used to tell the difference between autoflourescent portein light from light belonging from flourophore conjugated antibodies.

It is detected in Unstained WBC control.