NECyto 2016 follow-up

Thanks to all of you who attended the 2016 NECyto meeting this year, we hope you enjoy the t-shirts!  Jennifer Wilshire has shared her Forensic Flow talk and a quiz that has “Crimes of FACSing” in it that we didn’t get to during the meeting.  She welcomes you to use the slides and quiz in your own teaching and edit however you need.

Forensic Flow


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Fall Meetings Information

Plenty of educational and networking opportunities in Boston this fall!

Online registration is now open for this year’s NECyto Fall Meeting!  We will have 8 talks, lunch, and a vendor fair, returning to the Ragon Institute after our successful meeting last year.  Please find the speaker list, schedule, and additional information (including the link for online registration) here:

While you are in Boston/Cambridge, why not stick around for the weekend for some additional events?  The NERLSCD Meeting will be held from Oct 13 through Oct 15th
NERLSCD 2016 Registration is open!
If you are a manager or director of a core facility, you should consider attending the 11th annual NERLSCD meeting, to be held Oct 13-15, 2016 , in Boston MA.
The North East Regional Life Sciences Core Directors meeting (often called the NERDS meeting) is an outstanding regional forum for core facility administrators, directors, managers and staff to network with colleagues, to learn about biotechnology advances and their applications, and to discuss the challenges of implementing shared research resources.
Registration link:

Finally, the Biacore Users Group meeting will be held on October 14th at the Ragon Institute.  Learn about applications for SPR (Surface Plasmon Resonance) technologies. More details to follow, contact if you are interested.

We hope to see you in Boston this year!
The NECyto Organizing Committee

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facs pain

I experienced #10 the other day, and thought it presented a good opportunity to demonstrate the usefulness of spectraviewers and the importance of knowing your reagents. I’ve compiled a list of spectraviewers on this site, feel free to suggest additional links: (, but even then as I have lamented earlier, you can’t always compare your fluorochromes that are proprietary dyes from different vendors.  Biolegend has some cross-vendor spectra, but their selection is not complete (only a portion of the Live/Dead fixable dyes are shown).  The panel selection program Fluorofinder ( can show spectra for each of the dyes in their multi-vendor database, but don’t yet have a spectraviewer tool to view multiple fluors on the same plot (but that should be coming soon).

So to address the comment about fluors in the same channel, lets use BD Biosciences’s spectraviewer to look at the 2 dyes in question, and we’ll add in the next detector up that is used for BV510 on our system.  The colored bands are the default emission filters used on the system with a long pass in between to split the signal.facspain2

You can see that the 440/40 filter only captures just over half of the peaks of BV421 and Pacific Blue signals and catches the tail of spillover from BV510. This prevents you from having to change the filters around with every change in fluorochrome, but you can see that if you always run BV421, you might want to switch to something like 425/20:


This catches the whole peak of BV421  while avoiding the majority of BV510 spillover.  If you look at the spillover you get from BV510 into each of these filters there is a clear difference (same voltages for both plots):


And when you calculate the compensation matrix, the BV421-%BV510 drops from 30.6 for the 440/40 filter to 0.1% for the 425/20 at the same voltages.  Less spillover means less spread, so comparing compensated BV510 stained beads using either filter and keeping the gate in the same position for both plots shows:


This spread makes it harder to distinguish dim double positives, so if we look at single and double positive events, more double positives don’t resolve themselves from the single positives in the 440/40 setup (2.7% vs 0.7%).


(I ran the populations separately so I could tell how much overlap there was).

With so many reagents to choose from, and different vendors using different names for the same thing, even the experts can lose track, and new tools are always welcomed.  If you know of any programs, platforms, or websites that you find helpful in your flow cytometry research, please post it in the comments and I’ll add it to our links.  Be sure to make use of the existing tools to learn about the properties of your reagents, and talk to your core managers to learn about the setup for your particular systems to get the best results from your experiments.

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Flow Ornithology?


(That would be a pair of Buteo jamaicensis, but we have also seen a few Falco peregrinus and various members of the Laridae family…)

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Two quick meeting announcements to share for an old colleague, and don’t forget about our NECyto meeting on October 6th!

NERLSCD 2015 Registration is open!
If you are a manager or director of a core facility , you should consider attending the 10th annual NERLSCD 2015 meeting, to be held Oct 14-16, 2015 , in Burlington VT.
The NERLSCD meeting (often called the NERDS meeting) is an outstanding regional forum for core facility administrators, directors, managers and staff to network with colleagues, to learn about biotechnology advances and their applications, and to discuss the challenges of implementing shared research resources.
The early bird deadline for discounted registration and hotel rates is Sept 18th, so register soon!
NERLSCD is a regional chapter of the ABRF.

MetroFlow 2015 Meeting Announcement
Registration is now open for the MetroFlow 2015 meeting!
Join us on October 22, 2015 for another great full-day meeting, to be held at the NY Academy of Medicine (1216  5th Avenue , between 102 and 103rd St.)
We have a content-rich meeting planned, with confirmed speakers including:

  • Bob Balderas (BD Biosciences)
  • Chris Springs (University of Toronto)
  • Jennifer Wilshire (MSKCC)
  • Mark Edinger (Q2 Solutions)
  • Mark Fereshteh (Bristol-Myers Squibb)
  • Pratip Chattopadhyay (Vaccine Research Center, NIAID, NIH)
  • Teresa Hawley (The George Washington University)

Topics presented will include multicolor experimental setup and optimization, fluorescent proteins in flow, compensation and standardization, extracellular vesicle detection, sentinel gating, and high throughput analysis.For more information, please visit the MetroFlow website:
We hope to see you there!

The MetroFlow Steering Committee
Peter Lopez
Research Assistant Professor of Pathology
Director, Core Cytometry Facility, Office of Collaborative Science
NYU School of Medicine
540 First Ave Skirball 2nd floor Administration
New York, NY 10016
212.263.0635 (office)
212.263.5907 (lab)
NYUSOM OCS Core Cytometry Online

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NECyto 2015 registration link

Online registration for the 2015 New England Cytometry User Group annual meeting is now active:

This years talks:
“Alternative Aerosol Testing Methods”
Mike Waring/Mike Xie–Ragon Institute

“Automating Clinical Lab Analysis:  CD34+ Stem Cell Enumeration: A Software Perspective”
Bruce Bagwell/Beth Hill–Verity Software House

“Evolving Strategies and Technologies in Cell Sorting and Separation”
Joe Trotter–BD Biosciences

“Tunable laser technology for flow cytometry”
William Telford–NIH/NCI

“Targetable biosensors for high throughput drug discovery”
Alan Waggoner–Carnegie Mellon University

“Identifying and Quantifying Heterogeneity in High Content Analysis:  Application of Heterogeneity Indices to Drug Discovery”
Albert Gough–University of Pittsburgh

“The Use of Imaging Flow Cytometry as a Support Tool for the Traditional Flow Lab”
Scott Mordecai–ISXperts

We hope you will join us!
The NECyto Organizing Committee

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NECyto Fall Meeting 2015

Save the Date!
On behalf of the New England Cytometry Users Group Organizing Committee, I would like to announce that the Annual Fall Meeting will be on October 6th, 2015 from 8:00 am to 5 pm. We will have another great lineup of speakers! This year we will be changing venues, and will hold the talks in the Mark and Lisa Schwartz Auditorium of the Ragon Institute at 400 Technology Square in Cambridge, MA, and have a larger space for vendors and catering.

We hope you will join us!

New England Cytometry Users Group

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Getting Diva and Flowjo to play nice

Different export formats in Diva work differently in Flowjo, and Diva 8 has some changes from Diva 6 that I thought it was worth addressing…

We have Diva 6 running on our LSRs and Diva 8 on our SORP Aria. So in Diva 6, when you export as “FCS”, the file name is “Specimen_tube.fcs”, so when you load the file into Flowjo the file is identified by the filename (which is specimen and tube), and all parameters map correctly.

When you export as “Experiment” in Diva 6, the file name for the fcs files is a 5 digit number associated with the database in Diva (“34567.fcs”), and if you use these files in Flowjo the file name in the workspace is just the number (which you can get around by having the workspace display the tube name for the file so you can identify them) and if you display H and W parameters, they are switched—FSC-H is showing FSC-W and vice versa. Area is displayed normally.

NOW, in Diva 8 (not sure about 7, I didnt have it long enough to know), BOTH exporting formats (experiment or fcs) label the file “specimen_tube_###.fcs” where the number is the order of the files in the experiment layout, so you can’t easily tell the different export formats apart…

The standards for field labels for FCS 3.1 can be found here on page 10 of 34:
but this can get confusing because different software platforms can use different fields for different things, such as the $SRC field, or “source”, is used to store the “Specimen name” in Diva.

$FIL is for the filename.  For EXPERIMENT exported files in BOTH Diva 6 and 8, this is the number assigned to the file in the database, which you can also see in the D>BDDatabase>BDData folder.  For FCS exported files, $FIL is “Specimen_Tube name” in Diva 6, or “Specimen_Tube_###” in Diva 8. When you load a file that was exported from Diva 8 as an experiment, Flowjo will go to the $FIL field to display in the workspace, which in this case is just a number even though the name of the actual file is from the specimen and tube.

In the Flowjo preferences, workspace tab, “How should data files be named” you can choose to specify keywords instead of the default which uses the $FIL field. Choose “$SRC” (specimen) and “Tube Name” and as you choose each it will add it to the list below the drop down menu.  There is an option to “clear keywords” in the pulldown menu to clear chosen keywords. That will set the “name” in flowjo to display as specimen tube instead of filename, and FCS or experiment exported files will show up the same way, but keep in mind that Experiment exported files will STILL switch H and W signals.

Exporting in Diva
So this brings us to data management and exporting in Diva.  In Diva 6 on our LSRs, if the experiment isn’t that large I typically export as experiment, which creates a folder with the name of the experiment, and in that folder creates an xml file named after the experiment and then the fcs files which are all just numbered. Then I’ll export as fcs, which if done on its own will create a folder named after the experiment, but since that already exists from the experiment export, it just sticks the fcs files in that same folder.  The “experiment” exported files are numbers, and the “fcs” exported files are all named Specimen_Tube. You cant do this in Diva 8 because the file names for FCS or Experiment are the same, so you have to segregate them into separate folders and keep track of which is which.

Another local core facility (thanks Laura, who also helped me figure out some of the details of what was going on) will actually set the experiments to export as zip for all new accounts, so that the folders are kept separate that way by default (Diva will remember your choices from the previous export, so directory path, zipped, and in Diva 8 whether you want FCS 3.0 or 3.1).  You really only want to use the Experiment export in Diva, so zipping it is a good way to keep it apart, and when you export as FCS it will make a new, unzipped folder named after the experiment for your fcs files.

If anyone has any other solutions you would like to share, or clarifications if I’ve gotten some details wrong, please comment or email me!

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Einstein Express

We have a request form that our sort users have to submit to request sorting time, and they often have a copy that they have saved to re-submit, and often forget to update the date, it made me think of the old SNL skit:


if you haven’t seen it:

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Tracking settings

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:

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