Lecture 2 - Applications of Flow Cytometry

Question 1

List some general applications of flow cytometry

Answer 1

• Cell surface molecule expression • Intracellular molecules expression • Cell function • DNA content cell cycle analysis • Apoptosis • Antigen specific cell function • Cell sorting

Question 2

Describe how cell surface can be assessed with flow cytometry

Answer 2

Detection of CD4/8+ cells • anti-CD4 FITC • anti-CD8 PEFilters that detect each of FITC and PEScatter plots / histograms

Question 3

Describe how proliferation may be assessed

Answer 3

Stain: CFSE (Carboxyfluorescein succinimidyl ester)Stains intracellular proteinsDaughter cells will have exactly half the CFSE fluorescence than the parent celle.g. CD4 T cells • Naïve cells will stain the most brightly • After one round of division, the cells will fluorescent half as brightlyIn this way, we can assess the proliferation of lymphocytes

Question 4

Describe how antigen specific T cell responses can be measured using flow cytometry

Answer 4

Two methods: • Tetramer staining • Cytokine staining1. Tetramer structure • MHC (MHC I, II, CD1d) • Biotin • Peptide • FluorochromeFour of these subunits are held together with a streptavidin molecule2. The tetramer binds to T cells expressing TCRs specific for the MHC+peptide of the tetramer3. Cells are assessed using flow cytometry, population of cells of desired specificity can be counted

Question 5

Why must tetramers be used for assessing T cells of a given specificity?

Answer 5

A monomer would have too low avidity for the TCRA tetramer has increased avidity for the T cell, and thus will remain bound longer, so that the cells may be assessed with flow cytometry

Question 6

What is CD1d?

Answer 6

An MHC like molecule that loads lipids and glycolipids for presentation to NKT cells