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Multicolour Analysis

Multicolour Analysis - Blizard Institute

Human and murine platelets from whole blood can be immunophenotyped by the labelling of GpIb and GpIIb/IIIa to determine the presence of the respective clinical syndromes Glanzmann's Thrombasthenia and Bernard-Soulier syndrome and also the counting of platelet numbers.

Preparation of cell suspensions from solid tissues and cell cultures

Cell suspensions can readily be prepared from monolayers of cultured cells, e.g. human umbilical vein endothelial cells (HUVECs) by the dissociation procedures used when passaging cells (see below). They can also be prepared from solid mammalian tissues by disrupting cell-cell junctions and/or the interactions between cells and the 

There are a wide variety of commercially available fluorochromes conjugated and unconjugated to monoclonal antibody reagents which are used to detect a wide variety of surface antigens. The choice of fluorochromes is limited by the laser excitation sources available in your flow cytometer(s). The instruments available at the Blizard core facility allow most of the commercially available fluorochromes to be used with some limitations.

To detect intracellular antigens it is necessary to permeabilise the cell. Many reagents are available commercially to facilitate this.

The experimental design of a multicolour flow cytometry experiment has a number of issues that should be carefully considered including which fluorochromes to use, fluorescence minus one (FMO) technology, when to use and not to use isotype control antibodies, digital compensation and the use of single colour compensation beads for antibody fluorophores and the compensation issues involved with the use of annexin V, FPs, DNA and functional dyes in combination with antibody conjugates.

Introduction

Blue fluorescent protein (BFP ) was first isolated from the jelly fish Aequorea victoria, this weakly fluorescent version is maximally excited at 383nm and emits at 445nm. The greatly enhanced fluorescence of Green fluorescent protein (GFP) made up of 238 amino acids and 26.9 kDa or 9nm in length is almost maximally excited at 488 nm and emits at 509nm. The green fluorescence of GFP is produced when the aequorin molecules interact with calcium ions.

There are a wide variety of commercially available fluorochromes conjugated and unconjugated to monoclonal antibody reagents which are used to detect a wide variety of surface antigens. The choice of fluorochromes is limited by the laser excitation sources available in your flow cytometer(s). The instruments available at the ICMS core facility allow a most of the commercially available fluorochromes to be used with some limitations. The tables below show the fluorochromes and cell dyes and stains that can be analysed by flow cytometry at the BICMS flow cytometry core facility.

Lymphocytes - Thymocytes

Immunophenotyping of human and murine lymphocytes as well as murine thymocytes is a common application of multi-colour or polychromatic flow cytometry.

To detect intracellular phosphorylated antigens by flow cytometry it is necessary to permeabilise and fix a cell population in a particular manner. This is necessary not only for phosphorylated proteins but any to maintain any surface immunophenotyping of cells.

This form of analysis is probably the most common but there are number of considerations to be made about cell preparation. This should be discussed with one of the contacts.

Qdots and Fluorescence

Quantum Dots or Qdot nanocrystals in principle have similar fluorescent properties to the traditional organic based fluorescent dyes commonly in use today. Qdots are the size of proteins (up to a few thousand molecules) at 10-20nm diameter and are made up of a semiconductor core, (cadmium with selenium or tellurium) and a shell of zinc sulphide that improves the optical properties of the qdot. The Qdot also has a polymer coating and a layer of biomolecules, see figure below.

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