Flow+Cytometry+Questions+1-6-12

Contacted Beckman Coulter

I am using the Beckman Coulter FC500 flow cytometer instrument, but I am kind of new to flow cytometry. I have a few questions. What are appropriate voltages and gains to use to obtain a good SS FS dot plot for immune cells such as T cells and B cells. I just performed an experiment the other day, and I tried to adjust the settings to center most of the dots a little more. I ended up with the following settings: FS Volts 635 Gain 5.0 SS Volts 431 Gain 50.0

However, I have no idea if these settings are reasonable.

Also, I have a question about preparing my cells. I had some cells and I added a dye conjugated antibody to them. I let them incubate for about 15 min in the dark. I then centrifuged them because I was going to remove unreacted antibody-dye, and then resuspend the cells in PBS. However, I couldn't see very much of a pellet after centrifuging. I was very worried about losing my cells when I tried to remove the supernatent. Therefore, I didn't remove the supernatent. I just resuspended my cells in the same solution, and took this for flow cytometry. Would doing this cause any major problems? Would all of the unreacted antibody-dye be detected, or would it not be picked up since an unreacted antibody-dye has a very different size than a cell?

Also, what is the best way to centrifuge and remove the supernatent when the cell pellet and solution are in a 5 mL flow cytometer tube? Thanks for any information you can offer me!

Beckman Coulter?s Reply:

Thank you for your inquiry. May I offer these suggestions. Your laboratory must optimize the resolution of the leukocyte populations dependent on the application, prep technique and age of laser and pmts. There are many preferences of how the populations are displayed on a dual parameter/bivarite plot. With that in mind and without any data available the voltages that you have sent are higher than normal but not unusual. If you are new to flow cytometry my suggestion is to obtain an onsite visit of our Application Specialist or call Technical Support 800-526-7694 with the SID of your unit. Preparation of your sample for mab staining should follow the instructions for use (IFU); this information will give you the appropriate lysing, quick spin and/or washing methods necessary for optimize staining. Unstained or isotypic controls are useful for nonspecific and background staining. I hope this information is helpful. Regards.

Thank you,

Beckman Coulter, Inc. www.beckmancoulter.com