Basic flow questions - Need your help for these graphs and their use (Dec/04/2009 )

I am very much interested in learning the basics of flow cytometry, I found Partec Flow Cytometry presentations while I am browsing information about the flow cytometry instrumentation and graphs. I have few questions regarding the graphs that you have given in the presentation given below.
These information is given in page 10 and page 30.

http://www.mre.vghtpe.gov.tw/news/tables/p...w_Cytometry.pdf

What is the role of FSC vs Counts and SSC vs Counts in flow cytometry graphs? What information we can find using these graphs?
I will be very thankfull if you could pass on this information for my future.

Thanks a lot

Regards,

-Pls help-

CellSpecific.com on Dec 6 2009, 04:34 PM said:

When evaluating cells by flow cytometry, the FSC axis provides information about the size of the cell whereas the SSC axis provides information about the inner complexity of the cell such as the shape of the nucleus and the amount of cytoplasmic granules. One can use these information to specifically localize or focus evaluation of cellular markers of interest to a particular cell population based upon size (FSC) and granularity (SSC). The image shown below shows evaluation of smaller and less granular lymphocyte population (gated "highlighted" in red). Notice the larger and more granular neutrophil (PMN) population (not gated). The right panel image displays the level of those markers found on the "gated" lymphocyte population. Hope this helps.

Thank you very much for your information. I have one more question...

I am planning to perform immunophenotyping soon, I am using both primary and secondary antibodies for this analysis. Can I perform Immunophenotyping without taking PI as counter stain i.e by taking these graphs only.

FSC Vs SSC and FL1 Vs counts.

I will thankful ones again if u can reply for me..

Thanks.

-Pls help-

PI is not counter stain. It is stain to exclude dead cells. Those cells that take up the stain are all dead cells and give aberrant readings. So, you cannot be confident about your data unless you are already working with dead cells. Example, when I work with fixed cells for flow cytometry, I do not put PI as there is no meaning of that. However, there are other stains also that can replace PI if U really do not want to have PI.

-Nabi-