When a researcher is using the same panel for experiments run on different days, the temptation is to just use the same voltages as the first day. Due to instrument variations (fluctuation in laser power, variation in detector sensitivity due to heat and humidity, how long the instrument has been on, etc), this likely will not give you the same results. The only way to compare samples run on different days is to make sure the instrument is providing the same output for the same fluorescence signal on both days, to confirm that differences between data files are due to actual sample differences and not to instrument variation.
Keep in mind that staining consistency is also critical, as variation in your technique can lead to differences measured on the cytometer, and a good control for this is to run the same sample on each day and make sure they are consistent– so have a bank of PBMC aliquots from the same patient, and stain one with each experiment. If the instrument is consistent, and the sample is matched, then any variation would be due to changes in the staining and this will give you an idea of the consistency of your staining on different days, and how much of a change is actually significant.
With BD instruments, CST (Cytometer Setup and Tracking) in Diva can adjust voltages based on daily CST results (“Application Settings”), but you have to re-synchronize the settings for each new configuration, batch of CST or each new baseline for EACH user, which becomes challenging for a core facility with many users.
Another benefit of monitoring your instrument response for your experiment is that if someone has swapped filters on the instrument and neglected to restore them to “default”, it will usually be readily apparent in a large shift in your bead peak and hopefully shorten any troubleshooting you might have to do (“Gee, my pacific blue single stain has no signal, let me re-make the comp tube. Still didn’t work, oh wait someone swapped the filter…”).
Here is the writeup:
I’ve posted PDF’s of the talks that were presented at NECyto 2014 on October 2, 2014 in Boston, MA. They can be found here:
I would also like to announce a Business Skills Workshop for core managers that will be hosted by the University of Virginia, view the flyer here. Joanne Lannigan announced it during her presentation on successful core management, I’m sure it would be a useful tool in developing a successful core if you are able to attend.
Thank you to all of the attendees, vendors, and speakers at the 2014 NECyto meeting. We had over 120 participants and a great series of talks which we plan to post as PDFs in the coming week.
I’d like to announce an upcoming event tour presented by BD Biosciences, on October 28th in Farmington, CT, October 29th at the Ragon Institute in Cambridge MA, and November 4th in Houston, TX. There will be three seminars (“Introduction to Flow Cytometry”, “BD Accuri: Applications of Flow Cytometry in Cancer Research and Cell Biology”, and “Flow Cytometry as a Tool for Microbial Analysis”), followed by an instrument demo of the Accuri C6. Please view the flyer here.
and a flow related comic added to the Humor page…
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.