Data Analysis1

Lectures

Introduction
The flow cytometer
Data analysis
Immunofluorescence
Some clinical applications

Flow Cytometry Course

Data analysis - 1

In this lecture you will learn about analysing flow cytometry data files. You will be introduced to the concept of ‘gating’ as a means of selecting sub-populations of cells.

With the exception of the FACS Aria cell sorter from BD, the data files from all other current instruments are saved in a standard format, FCS 2.0, so that they can be read by any of the software supplied for this purpose. The DiVa software supplied with the FACS Aria uses FCS 3.0.

You have been supplied files of flow cytometric data recorded from during an analysis and saved as ‘listed’ data files. You will need to use an appropriate computer program to analyse them. You should read the comments related to this in the Introductory remarks (here).

Introduction

All flow cytometers have a computer associated with them. The computer program controls the cytometer during data acquisition. It is used to:

·	select the parameters for measurement;

·	select area, width or height on different parameters (for pulse processing);

·	adjust the voltages on the PMTs;

·	adjust the gain settings on the amplifiers;

·	select logarithmic or linear amplification;

·	select and adjust the threshold (discriminator) settings;

·	adjust the settings for colour compensation (see Lecture 4);

·	select histograms and cytograms for display;

·	draw regions and set gates (see below) to be used during data acquisition.

If the flow cytometer can sort cells, the computer controls the sorting process.

The computer program can also be used to analyse data subsequent to its acquisition. Off-line analysis is also useful for the preparation of illustrations for publications, lecture slides, etc.

It is convenient to have a program for analysis of listed data files on computers in other locations. There are a variety of programs supplied for this purpose; some of them are sold commercially, others are free. Whatever the program used, the principles of data analysis are the same.

If you are using WinMDI as your analysis program, go here.

If you are using FCS Express Reader, go here.

Otherwise, continue here.