Hello everyone.
I am doing a multicolor flow cytomtery experiment. My cells are bovine lymphocytes and I satin with 4 colors including; FITC, PE, APC and a violet kit proliferation dye. I am in step one of creating my compensation controls and I use the canto machine. I had my unstained and single color tubes. when I adjust the voltage using the unstained, i miss lots of my positive population in my single color tubes and if I adjust my voltage individually at each single color, I miss lots of the negative population ( I apply the adjusted voltages again to the unstained to keep consistency throughout the experiment). Any clues how to adjust the negative and positive populations?
Thanks all
Initial question, do you use a voltage-setting QC program on your machine (such as BD DIVA CS&T?)
Agreed. Sounds like a CS&T calibration is needed to set the appropriate linear range for each parameter. Is this happening for all your compensation control tubes? Or just one in particular?
Setting voltages based on the position of the unstained cells is a traditional practice, but probably isn't best practice for your data. If your cells are off-scale (on the top end) you will end up with compensation (analysis) problems, so you should definitely reduce your voltages so all your events are on scale.
In terms of 'missing a lot of the negative population', this isn't exactly what happens. The negative population doesn't disappear from the file. Even if your voltage was 50V you would still keep all the events that were triggered on the cytometer, but they might stack up on the lower axis meaning that it looks like they have disappeared off the low end. What can happen if the voltage is too low, is the you can get low-fluorescent events collapsing into the negative, and so you 'lose' dim events. However, it is (usually, depending on your question) more important to have your events on scale (at the top end), rather than have your dim populations separate from negative.
So this is what I would do:
1. Use the CS&T program to set your voltages (if you have it, otherwise see the link below)
2. Examine all your fluorescent parameters and reduce the voltage for any that have events off scale
3. For the others, leave the voltage as it has been set by CS&T
There is some more detail about setting voltages (partly based on unstained cells) at the following link, this may be helpful for you:
https://www.bdbiosciences.com/documents/BD_FACSDiva_Stndrd_App_Setup_TechBulletin.pdf
It could be a simple problem of whether your cells express the markers at high levels; if they do not, using these cells as your positive single stains may not be suitable. Try checking the literature if the markers are commonly and highly expressed and try using compensation beads as a guide to adjust the voltage instead.
- Badges
- Science method