Hey everyone, can any one help me the recommended values for different parameters listed under flow cytometer control including, discr, volts and gain for FSC, SS, FL1,2,3 ..6? Should the values vary based on cancer cell lines under analysis? Thanks, your help is appreciated!
Hi There,
Voltages are a hard thing to guess as they depend on a variety of different variables which are different between machines. So I could tell you what my machine is set at and that will not work for you. It depends on which machine, what filters, laser strength, the size of cell and what the surface of the cells looks like, and even antibodies use and how much signal for each antigen the cell has.
Despite that there are a few things you can control for.
Generally have plenty of unstained cells and run your machine slowly for set up. For your FSC and SSC you want your population within the middle of the plot. Be careful about cells cramped around the edges.
For colours, set your unstained at a known setting. Depending on your machine, this could be 0 to 10e1 or 0 to 10e3. If you set to a known negative value, everything above that is positive. I have had to set this lower on very bright signal so always check on a bright signal too.
Each cell type will have a different level of auto fluorescence. You should have an unstained for each type to make sure you have set the machine up right. Culturing will change this as most media has Phenol Red in the media. You can buy the Phenol Red free media but I don't bother.'
Also have a chat to your flow cytometery technician, they are great to get to know
Hope this helps
Regards Bianca
Thanks Bianca, that's really helpful information. I will try to include the unstained control in my next experiment. the main issue I faced in this experiment is that even if I have enough number of cells which are adequately stained, fixed in ethanol and incubated for 40min at 37oc, I couldn't get well defined peak using FACS for cell cycle analysis. I have tried this twice with MCF-7 breast cancer cell line and MV4-11 leukaemia cell line. My instrument is Beckman culter. Thank you once again for your suggestion.
We had this issue with cell cycle. We actually ended up analysing our daya on a Beckman Dickson machine as it had a better laser filter combo. Ask your flow core they may suggest another machine.
Also put you sample in a larger volume and try running it slower sometimes fluidics will throw out signal making broader peaks.
Hope this helps
Your unstained sample will also need to be fixed too.
Regards B
Bianca, that's so lovely suggestions. but, please could you clarify for me what you mean by ""put your sample in a larger volume." because, usually what I do is stain the samples with 200uL propidium iodide, incubate and then add 200uL PBS right before running it with FACS. Thanks a lot!
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