Hi all
I had gotten some feedbacks from some of our fellow course-mate that my entries are a little too technical and very hard to understand. So I decided to skip the technical stuffs and go straight in to the topic with simple explanation.
So feel free to ask me any questions regarding any parts that you do not understand in my entry.
Simple 4 steps explanation of how an experiment with the Fluorescent Activated Cell Sorter (FACSAria), also known as a flow cytometers, works:
1) Load the samples onto the FACSAria
2) Data will be acquired
3) Set gating to select out the cells of interest
4) Record the data
Flow cytometry is simply just to analyze the data acquired from cell population that was loaded onto the FACSAria. It analyze by detecting and identifying the cell population of interest that were set aside differently from rest of the cell populations.
Some examples of how this could be done are through the uses of cell markers and certain dyes, like fluorescent-coupled antibodies or fluorescent dyes or fluorescent proteins.
Example of Fluorescent coupled antibodies:
1) Antibodies against CD44 cell marker, coupled with PE fluorochrome
Note: PE stands for phycoerythrin
Example of Fluorescent dyes:
1) Hoechst 33342
Example of Fluorescent proteins:
1) Green Fluorescent Protein (GFP) via cell transfection
Okay, that’s all to my introduction to the flow cytometers and I will stop any further complex explanation here. Hope everyone could have a rough understanding of what flow cytometry is about.
Many thanks
Quan Jun
TG02
Group 08
08 September 2008
Double posting! (1st part: Simple Introduction to Flow Cytometry)
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2 comments:
September 13, 2008 at 1:06 PM
Hi Quan Jun,
1) What information does the data generated from the flow cytometer tells you?
2)Flow cytometer entails the use of dyes for different applications? What are the factors to consider when choosing a suitable dye?
THanks!
From: Benjamin Ma
CLass: TG01 0606181F
September 15, 2008 at 8:50 AM
To Benjamin:
1) The information actually depends on the user experiment design and the fluorochrome used.
3 basic information flow cytometry can provide researchers are:
- % of the cells of interest (if it's from a heterogenous population)
- % of cells expressing the cell marker of interest (often used to identify the cell of interest)
- Viability of the cells
2) Not really different applications, but rather, the use of different dye combination enables the identification and isolation (if the researcher wishes to) of the cell of interest.
Summarizing it all (the basic):
The use of dyes enable you to find your cell of interest and it's viability count and isolating them via sorting (if you intent to grow those cells).
Two main factors to consider when chossing a suitable dye are:
i) reagents
- can you find your dye commercially prepared?
ii) instruments
- do you have the necessary instrument compatible with the dye you want to use?
- Example: do you have an excitation light source at 405nm wavelength that is required for the excitation of Alexa Fluor 430?
I could probably add more to the list of answers, but this is the basics you should know. You will just get more confuse, with the more I explain.
For more information on the factors to choosing a suitable dye, you could go to:
http://www.med.umich.edu/flowcytometry/InitialTraining/lessons/lesson8/index.htm
Hope I didn't give you a headache reading =)
Many thanks
Quan Jun
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