There persists a misconception that if things are measured in the same channel, they are the same color or “close enough”. The example that I’ll use to illustrate the potential problems with this was a panel that used BV510 and V500 as the labels for the dump channel markers, and Live/Dead Aqua for viability, which are all measured off of 405 excitation and a bandpass around 525/50. You can see in the image below that they are all slightly different:
http://www.biolegend.com/spectraanalyzer A quick aside:
On our system, we have the following detectors on our violet laser. Shown are the uncompensated signals from the 3 different dyes on single stained beads.
If you compensate based on V500, you can see the other parameters are not properly compensated in all channels:
Compensating based on BV510:
And Live/Dead Aqua:
The cytometer has no way to tell WHICH dye is generating the signal, so it will not be able to correct the signals properly, but will just apply whatever values your matrix contains. The actual matrix values for the different dyes:
If you still decide to mix fluors, in this case (viability combined with a dump channel) I would use the Live/Dead dye to determine compensation. ALL cells will be stained with the L/D dye (dead cells will be brightest) so errors in compensation will affect your populations of interest. For a dump channel you are keeping the negative cells so even if compensation isn’t quite right, it should not affect your cells of interest.
Note that a similar situation can occur if you use a tandem dye for your dump channel–the tandem for each different antibody may be slightly different, so your compensation may not be correct for all markers.
We have posted the talk titles for our speakers at this years NECyto Fall Meeting being held on October 2, 2014:
Registration deadline is September 26th.
And if you are in town the day before, come across the river to join the Ragon Institute for a talk presented by Biolegend’s Kelly Lundsten (view flyer):
“Characterization of Chemokine Receptor Antibody Function and Human Chemokine Receptor Expression”
And another new entry to the humor page, in a galaxy far far away…
For those of you from New England, or if you are planning to come for the New England Cytometry Fall Meeting on October 2, come a day early!
There will be a presentation by Biolegend on Wednesday, October 1, 2014 from 9:30 to 11am in the Mark and Lisa Schwarz Auditorium at the Ragon Institute, 400 Technology Square in Cambridge MA:
“Characterization of Chemokine Receptor Antibody Function and Human Chemokine Receptor Expression”, presented by Kelly Lundsten. Topics will include 13-15 color flow cytometric assays to examine chemokine receptor expression of NK, T-cell, B-cell, macrophage, monocyte, and dentritic cells and the challenges related to such analysis.
View the flyer here:
New England Cytometry’s Annual Fall Meeting will be held in Boston on October 2, 2014. Attendees can register online here:
The speaker list has been posted to
And finally, visit the humor page to learn about FMOs from Captain Picard.
New England Cytometry’s Annual Fall Meeting will be held in Boston on October 2, 2014. We are assembling another great list of speakers and will announce the lineup soon, but attendees and vendors can register now by going to the following link:
The speaker list will be posted to
We hope you will attend!
I’ve also posted a new edit of my Spinal Tap post in the Humor section. Other posts in the works.
The New England Cytometry Users Group would like to announce the date for our annual Fall meeting. It will be on October 2nd from 8:00 am to 5 pm at the beautiful Starr Center at the Schepens Eye Research Institute in Boston, MA. Details will be posted here, or you can navigate to the page through the “Meetings” tab above. We hope to see you there!
A new job has been listed on the Job Board.
Finally, I’ve collected the published humor posts, and added a few new ones, to the Humor page.
–Thanks to Stephen Kwok at the Tufts Laser Cytometry Flow Core at Tufts Medical School for conducting a flow cytometry salary survey last year, the results were posted to the Purdue list but have since been removed, so we are posting it here for anyone that is interested. I’ll also link it from the “Job Board” page.
2013 Flow Cytometry Survey
–There is a new category in the menu bar for “Humor” so that the feed does not end up with jokes overwhelming the “scholarly” posts or announcements. Feedback appreciated!
For those of you that grew up with Sesame Street:
Three of these things belong together
Three of these things are kind of the same
Can you guess which one of these doesn’t belong here?
Now it’s time to play our game (time to play our game).
Click here for the answer!