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Flow Cytometry Data Analysis

               All flow cytometers are connected to a specialized computer in which a specialized flow cytometry software is installed.. When data are acquired, the software controls the cytometer and helps to select the number of required parameters, helps to select different parameters such as area, height and width, adjusts PMT settings, gain settings, amplification in linear or logarithmic, types of plots to display (Dot, Density, Contour or Histogram Plots), helps in adjusting the threshold and most importantly, it helps in drawing gates or regions around the population of interest based on their common characteristics such as side scatter (granularity), forward scatter (size) and fluorescence marker expression (fluorescence channels).
The main intention of this article is to explain "How to analyze a flow cytometer data, what are the types of data, how to represent and what are the simple steps in presenting a effective data.. 
And at the end of this article, Demo has been given for analyzing a flow cytometry data.

Before we begin, lets have a brief look on actually flow cytometry data looks like.




 Flow Cytometry Types of Data Plots and Types of Gating (Regions): There are mainly 4 types of data plots: Dot Plot, Density Plot, Contour Plot and Histogram Plot. Example of these is shown in the figure below. 




 Types of Gate (or Regions) to identify a cell population of interest:Polygonal Gate, Rectangular Gate, Ellipse Gate, Region Gate and Quadrant Dot. Example of these is shown in the figure below.

When you plan for a flow cytometry experiment, its a very good idea to have the knowledge of approximate size of cell, cellular characteristics (physical and chemical), fluorescence and conditions for the cells, so they can stay alive or fixed during an experiment.. importantly, autofluorescence of cells should be considered as this helps in identifying the real fluorescence of the cells. Here is an example below.
Consider you have a mammalian cell line, for example HeLa cells and you transfected them with the plasmid containing Green Fluorescence Protein. That means, HeLa cells will emit Green Fluorescence Protein (GFP) when you excite them with the 488nm Blue Laser. and to understand the mean and median fluorescence intensity.

(Basic analysis of GFP fluorescence of Transfected HeLa Cells)
Steps to Analyze or Record GFP Fluorescence of HeLa Cells:

 1) Prepare single cell suspension of HeLa Cells as per the guidelines. (Preparation of HeLa Cells for Flow Cytometry).

 2) In this case here, you will have two sample tubes. (Cells suspended in PBS or DPBS).
    a) Unstained or Not Transfected: (HeLa Cells with No GFP).
    b) Stained or Transfected: (HeLa Cells with GFP).

 3) First, Plot these data plots on the cytometry software.

4) Run the unstained sample tube, set the PMT and Gain Settings, then you will see this data.. Record at least 10,000 to 20,000 events for analysis.


 5) Now, Run the GFP Sample Tube, (Do not change PMT or Gain now), then you will see this data. here also, you have to record at least 20,000 events for analysis.

Did you the notice difference between unstained and GFP data?? Did you see the change in histogram plot and change in fluorescence shift??


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