Reply to Han Yang question from "Basic Introduction to Flow Cytometry"

Han Yang:

With regards to your questions, it would be understand best with pictures, so I decided to post a new entry for your questions, hope you don't mind.

1) 2 things:

1. One of the most important precautions to take note is to make sure there are as few cell clumps as possible. As this will clot your flow cytometer, where the troubleshooting will make you seriously regret not removing the cell clumps if you didn’t.


2. The next precaution relates more to the experiment design, if you want to isolate and sort a certain cell, you have to make sure you don’t have a lot of other unwanted cells in your sample (eg. RBCs, etc) or you are going to spend a long time sorting out the cells of interest due to very low % in your sample.

2) Lolz. I don’t mind sharing how the sample is load into the flow cytometer, you just place the sample tube on the “tube holder” on the “loading port” and load into the “sample injection chamber” of the flow cytometer by clicking on the "load" button on the "acquisition dashboard".
(Refer to figure 3 below for sample of "acquisition dashboard")

3) They are just some isotonic buffer solutions (eg. 1x PBS) that keep the cells structure intact for flow cytometry analysis. Some places even uses high grade DI water.

References:

• www.accuricytometers.com/customer_support/faqs/#faq_15
• www.fhcrc.org/science/labs/fero/Protocols/Flow.html
• www.healthcare.uiowa.edu/corefacilities/FlowCytometry/protocols/tips/index.htm

4) Regarding the principle, I am not sure what principles are you talking about, but let me just show you the picture of how the lasers are focused onto the optical lens (also known as focusing lens):

Other than that, I think it’s best if I don’t touch on the principle too detailed as it relates to a whole new subject on physics (lasers). Let’s not make the learning confusing, okay? =)

Note: The display of some acquisition dashboard might vary a little, but they all basically have the same functions.

Hope my explanation is clear and help in your better understanding! =)

Quan Jun