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New in Flow cytometry! I need your help please!


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#1 Butterfly's Hull

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Posted 25 July 2009 - 03:30 PM

Hello,

I'm doing my Msc research in Microparticles in Cancer. These Microparticles are cell membrane fragments released upon the cell activation or apoptosis. They are normally exist in the blood, but if they elevated in the circulation make some diseases especilly Vascular diseases. Lately they are investigatd in relation to pancreatic Cancer.
So the aim is to stimulate Cancer cells to release Micropatricles in response to Hypoxia, and then characterise them by Flowcytometry. So I still have questions about FlowCytometry. LIKE Why do we use NEGATIVE CONTROL? Why do we use positive control? What do they mean by Antigens in this case because I know that Antigens are some weird stuff in th body, so are they the Microparticls? Why do we need to use the fluorecense...I'm still confused. PLEASE any one has any handy information let me know. ;)

Thanks a lot in advance, and I highly appreciate it!

#2 illuminated

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Posted 26 July 2009 - 02:47 AM

Hi!

You cannot expect us to explain to you all the basics of flow cytometry. Start with this introduction: http://www.invitroge.../Tutorials.html and later I'll give you another useful link. Here is a forum discussion where you can find links to a very good flow cytometry book that is really worth to be read: http://www.protocol-...?showtopic=5967
Use Google to find answers to your questions (and also wikipedia!). To get your degree you are supposed to learn to find information autonomously! If afterwards you still don't understand something, than create a specific topic on your particular questions in this forum.

Regards

illuminated

#3 Butterfly's Hull

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Posted 26 July 2009 - 10:48 AM

Hi!

You cannot expect us to explain to you all the basics of flow cytometry. Start with this introduction: http://www.invitroge.../Tutorials.html and later I'll give you another useful link. Here is a forum discussion where you can find links to a very good flow cytometry book that is really worth to be read: http://www.protocol-...?showtopic=5967
Use Google to find answers to your questions (and also wikipedia!). To get your degree you are supposed to learn to find information autonomously! If afterwards you still don't understand something, than create a specific topic on your particular questions in this forum.

Regards

illuminated


Thanks a lot illuminated for your reply. I know how to find information, and I read a lot about flow cytometry online, and some books. But I had some questions that's why I asked. I put my questions already in my first post. I read most of topics in the fourm just to understand the concept of flow cytometry perfectly. As you know analysis of th results is not that easy for new student who is not native speaker. I learned American English, and my supervisor is British. I find his english and his accent tough for me. I'm trying to do my best to understand it.
I'm trying to understand now what negative control means, and how to understand my results. I already read a lot about the flow cytometry and my topic. I hope you understand my situation. Thanks again and appreciate your help and advice.

#4 Stephan

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Posted 27 July 2009 - 12:00 AM

Hi,

Firstly I do not consider myself any sort of expert but hopefully I can give some insight from my experiences.

Every time you do some sort of FACS analysis you must calibrate the machine (over an above usual bead calibration). I usually work with apoptosis and for this you need 4 vials which you have induced apoptosis. (these are your positive controls)
1 vial with the one anti body
1 vial with the other anti body
1 vial with both anti bodies

1 vial with no stain (negative control to check for auto-florescence)
The flow cytometer needs to be adjusted for your specific experiment with these positive controls.

The idea of positive and negative controls can get very confusing depending on your actual experiment. With yours, you are only looking for a specific micro particle. your positive control would have to be a soln where you are sure you have the said particle in. When this is run a near 100% confidence should be achieved.

This gives you a target or an area on your graph that you now know that only those specific microparticles exist. A negative control would be a soln which has none of the previous mentioned things and thus must prove that there is no natural occurrence of these particles.
Just don't forget about the whole array of further controls which you need. Such as normal controls and experimental controls - if you are unsure about which controls you should use, try think of possible reasons your results could not be due to what you originally hypothesized and then develop a control for it.

As for your other questions such as what is an antigen, Generally speaking an antigen is the thing which induces the production of antibodies in the body but with flow cytometry we are referring to anything the antibody binds to as the antigen. Thus you get antigen specific antibodies for your microparticle or cancer cell - which ever you are interested in.
If you don't know why you need to use fluorescence then you must read more on flow cytometry - this is the major mechanism of how it works.

Hope this gives you the right direction.

#5 illuminated

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Posted 27 July 2009 - 01:42 AM

Thanks a lot illuminated for your reply. I know how to find information, and I read a lot about flow cytometry online, and some books. But I had some questions that's why I asked. I put my questions already in my first post. I read most of topics in the fourm just to understand the concept of flow cytometry perfectly. As you know analysis of th results is not that easy for new student who is not native speaker. I learned American English, and my supervisor is British. I find his english and his accent tough for me. I'm trying to do my best to understand it.
I'm trying to understand now what negative control means, and how to understand my results. I already read a lot about the flow cytometry and my topic. I hope you understand my situation. Thanks again and appreciate your help and advice.


I cannot believe that you read a lot about flow cytometry as your are asking very basic questions. And I also don't believe that you tried to find the answers to your questions online. I just looked in Google for "flow cytometry control positive negative" and one of the first results was this one: http://www.abcam.com...ry_controls.pdf It's not hard to find at all.

What an antigen is belongs to basic knowledge that you can find in every cell biology and immunology book and even in wikipedia: http://en.wikipedia.org/wiki/Antigen The first sentence is:

An antigen (Ag) is a substance that prompts the generation of antibodies


So whatever you want to detect in your cells with an antibody will be an antigen.

The sense of using fluorescence is a basic concept of flow cytometry! Flow cytometry is based on fluorescence, so I don't understand your question about why to use it. Do you have any other suggestions what you could use for your purpose instead of fluorescence? I mean, you could use for example western blotting or 2D electrophoresis for protein detection, than you don't need fluorescence, but you said yourself that you want to use flow cytometry.

And I am sorry to say it but the English knowledge is not an excuse for not looking for proper information. I am not a native speaker either and I also learned American English at school and did my MSc in the UK but reading literature does not depend on the particular dialect and your supervisor is anyway not supposed to explain you everything as you are expected to find information yourself.

#6 Butterfly's Hull

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Posted 31 July 2009 - 05:12 PM

Hi,

Firstly I do not consider myself any sort of expert but hopefully I can give some insight from my experiences.

Every time you do some sort of FACS analysis you must calibrate the machine (over an above usual bead calibration). I usually work with apoptosis and for this you need 4 vials which you have induced apoptosis. (these are your positive controls)
1 vial with the one anti body
1 vial with the other anti body
1 vial with both anti bodies

1 vial with no stain (negative control to check for auto-florescence)
The flow cytometer needs to be adjusted for your specific experiment with these positive controls.

The idea of positive and negative controls can get very confusing depending on your actual experiment. With yours, you are only looking for a specific micro particle. your positive control would have to be a soln where you are sure you have the said particle in. When this is run a near 100% confidence should be achieved.

This gives you a target or an area on your graph that you now know that only those specific microparticles exist. A negative control would be a soln which has none of the previous mentioned things and thus must prove that there is no natural occurrence of these particles.
Just don't forget about the whole array of further controls which you need. Such as normal controls and experimental controls - if you are unsure about which controls you should use, try think of possible reasons your results could not be due to what you originally hypothesized and then develop a control for it.

As for your other questions such as what is an antigen, Generally speaking an antigen is the thing which induces the production of antibodies in the body but with flow cytometry we are referring to anything the antibody binds to as the antigen. Thus you get antigen specific antibodies for your microparticle or cancer cell - which ever you are interested in.
If you don't know why you need to use fluorescence then you must read more on flow cytometry - this is the major mechanism of how it works.

Hope this gives you the right direction.


Thanks a lot Stephan. I highly appreciate the time you spent to explain to me. I did went thru all about flowcytometry and now I feel much better. I understood about the floursence. I really got good idea from your experiemnt. I dod lately the flowcytometry analysis, but I'm still a little confused to how interepert the data. I'm trying though. I read a lot, and hopefully this will help me. Thanks again!!!! I wish you all the best in your studies!!!

#7 Butterfly's Hull

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Posted 31 July 2009 - 05:28 PM

Illuminated I don't lie. Thanks though!


I know this information about the Antigens, but I meant what It means in Flowcytometry. Stephen said really good stuff about it, and actually that all what I needed to know about it. Also I understood the fluoresence idea.



Actually I didn't accept the last words. Please Illuminated don't think that people have same your circumestances. Maybe yours were good, and you didn't have any troubles that affect your study path. Or maybe you studied continously. So please don't be judgmental since I'm not obligated to tell you my life story. If you have good information that benefit me that would be great, otherwise I don't want to hear such words please. I know myself I'm hard worker and new stuff need time to get it. No one was born educated, Maybe you!!!!!! Sorry to say this and thanks for your time.

Regrads

Edited by Butterfly's Hull, 31 July 2009 - 05:29 PM.





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