It's time to publish your exciting results, but how should you describe the instruments you used?


As instruments and experimental complexity develop, the requirement for precise information about the details of your experiment is essential.
Some journals stipulate a minimum requirement for information about your microscopy experiments (e.g. these instructions from Nature Publishing), and in 2008, the International Society for Analytical Cytometry (ISAC) published the MIFlowCyt standard - a standard outlining the Minimum Information about a Flow Cytometry experiment that is required for another investigator to be able to repeat your techniques and endeavours.

To assist with your write up, we have compiled all the specific details of our instruments in a journal-style format.

In addition to these details, you may be called upon to support your decisions about experimental controls and techniques. In the References section you will find a collection of publications describing particular controls and techniques that often require justification and supporting evidence.

If you are planning to acknowledge the Detmold Family Cytometry Facility in your thesis or publication (thank you!) our full affiliation details are:

Detmold Family Cytometry Facility, Hanson Institute, SA Pathology.

If you have any questions, or need other information, please don't hesitate to get in touch with us.

Cytometers

FACS AriaII (sorter)
...Becton Dickinson FACSAria II using FACS Diva Software version 8.0 (BD Biosciences, San Diego, CA, USA) equipped with 405 nm (30mW), 488 nm (20 mW) and 633 nm (20nW) lasers.
Be sure to mention the ACDU (automatic cell deposition unit) if you performed single cell sorting into plates.
For specific details about collection optics, please speak with Detmold Staff, we can refer back to the configuration details at the time of your experiment.
Astrios (sorter)
...Beckman Coulter MoFlo Astrios EQ High Speed Cell Sorter, using Summit Software version 6.2.4.15830 (Beckman Coulter, Miami, FL, USA) equipped with 355 nm (100mW), 405 nm (55 mW), 488 nm (150 mW), 561 nm (200 mW) and 633 nm (100 mW) lasers. The cytometer is enclosed within Baker SterilGuard BSL Class II Biosafety cabinet (The Baker Company, Sanford, Maine, USA)
Be sure to mention the CyCloneTM (automatic cell deposition unit) if you performed single cell sorting into plates or onto slides.
For specific details about collection optics, please speak with Detmold Staff, we can refer back to the configuration details at the time of your experiment, alternatively, you can extract the specific details from the FCS File header yourself.
Note: there has been an upgrade to the forward scatter detection on the Astrios to Astrios EQ (January 2016) – forward scatter is now detected through 1 (or 2) PMTs instead of a photodiode, which gives greater sensitivity and better signal to noise resolution of particles. If your assay required specialised FSC detection (e.g. small particles) you will need to mention this component. If you are unsure about the settings used for your assay, please ask.
Altra (sorter; decommissioned)
...Beckman Coulter Epics Altra HyperSort, using Expo MultiComp Software version 1.2B (Beckman Coulter, Miami, FL, USA)
The sorter was equipped with 2 lasers.
Innova I90 (Coherent), tuned to 640nm or 561nm with the laser power usually set to 100mW.
Innova I300 Multiline laser with beam splitter, UV split off to 488nm with the laser power usually set to 100mW
Be sure to mention the AutocloneTM (automatic cell deposition unit) if you performed single cell sorting into plates
We no longer have this instrument. It was actively used until June 2013. For specific details of the optical configuration used, please contact Detmold Staff.
LSR Fortessa (analyser)
...Becton Dickinson LSR Fortessa Special Order Research Product, using FACS Diva Software version 8.0 (BD Biosciences, San Diego, CA, USA) and equipped with 355 nm (20 mW), 405 nm (50 mW), 488 nm (50 mW), 561 nm (50 mW) and 633 nm (40 mW) lasers.
For specific details about collection optics, please speak with Detmold Staff, we can refer back to the configuration details at the time of your experiment.
Gallios (analyser)
...Beckman Coulter Gallios, using Gallios Cytometry List Mode Data Acquisition and Analysis Software version 1.2 (Beckman Coulter, Miami, FL, USA), and equipped with 405 nm (40 mW), 488 nm (22 mW), 561 nm (21.5 mW) and 638 nm (25 mW) lasers.
Note: if you used the 561nm laser, you should mention that it is collinearly aligned with the 488nm laser.
For specific details regarding the lasers and optical filters associated with each laser that are in use on the cytometer, please see Gallios Fluorochrome Options.xlsx (on the instruments page).
FC500 (analyser; decommissioned)
...Beckman Coulter Cytomics FC500, using CXP Cytometry List Mode Data Acquisition and Analysis Software version 2.2 (Beckman Coulter, Miami, FL, USA) and equipped with 488 nm and 640 nm lasers.
Unfortunately laser-power information for this cytometer is unavailable.
We no longer have this instrument. It was actively used until June 2013. For specific details of the optical configuration used, please contact Detmold Staff.

Microscopes

LSM 700
...Carl Zeiss LSM 700 Axio Observer.Z1 confocal microscope using Zen 2011 - Black Edition, version 8.1.5.484 (Carl Zeiss Microscopy, Jena, Germany) equipped with 405 nm (5 mW), 488 nm (10 mW), 555 (10 mW) and 639 nm (5 mW) lasers.
Objective specifications.
LSM Cal. 5x/0.16 (calibration lens)
PlanApo 10x/0.45 DICII
PlanApo 20x/0.8 DICII
C Apo 40x/1.2 W DICII
C Apo 63x/1.2 W DICII
PlanApo 63x /1.4 Oil DICII
If you used the incubation chamber, mention it along with the CO2 and temperature control you used.
Do not forget to include the details of your image processing software.
LSM 800
...Carl Zeiss LSM 800 Axio Observer 7 confocal microscope using Zen 2.3 - Blue Edition (Carl Zeiss Microscopy, Jena, Germany) equipped with 405nm (5mW), 488nm (10mW), 561nm (10mW), 640nm (5mW) lasers.
Objective specifications.
PlanApo 10x/0.45 DICII
PlanApo 20x/0.8 DICII
PlanApo 40x /1.3 Oil DICII
PlanApo 63x /1.4 Oil DICII
If you used the Airyscan mode be sure to mention that in your write up. Airyscan mode with the 63x Oil lens results in 1.7x greater resolution
If you were assessing transmission light you would have used the ESID detector
Do not forget to include the details of your image processing software.
LSM 710 two-photon
...Carl Zeiss LSM 710 two-photon microscope using Zen 2011 (Black Edition) version 7.0.4.0 (Carl Zeiss Microscopy, Jena, Germany) equipped with 458 nm, 488 nm, 514 nm and 633 nm lasers.
The LSM 710 is also equipped with a Tuneable Mai Tai DEEPSEE Ti:Sapphire multiphoton laser. If you used this laser, stipulate (in nm) the frequency to which it was tuned (tunable range is between 690-1040nm)
Objective specifications:
*LSM Cal. 5x/0.16 (calibration lens)
*PlanApo 20x/0.8
*PlanApo 20x/1 DIC
*PlanApo 40x/1.0 DIC
*PlanApo 63x/1.4 Oil DIC
If you used the incubation chamber, mention it along with the CO2 and temperature control you used.
Do not forget to include the details of your image processing software.
Bio-Rad Radiance 2100 (decommissioned)
...Bio-Rad Radiance 2100TM confocal microscope (Bio-Rad Laboratories, CA, USA) attached to an Olympus IX70 inverted microscope with LaserSharp 2000TM software version 5.2 (Bio-Rad Laboratories) for image capture, and equipped with 488 nm (Argon-ion), 543 nm (HeNe) and 637 nm (red diode) lasers.
Objective specifications:
*UPlanApo 10x/0.40 ∞/0.17 (Olympus)
*UPlanApo 10x/0.40W ∞/ (Olympus)
*LcPlanFl 20x/0.40 ∞/ (Olympus)
*UApo/340 20x/0.70W /0.17 (Olympus)
*UApo/340 20x/0.70W /0.17 (Olympus)
*UApo/340 40x/1.15W ∞/0.13-0.25 (Olympus)
*UPlanApo 60x/1.20W (Olympus)
Do not forget to include the details of your image processing software.
In active service until 2013.

Software

FCS Express version 3
(De Novo Software, Los Angeles, CA, USA)
FCS Express for Flow Cytometry Version 4
(De Novo Software, Los Angeles, CA, USA)
If you used the cell cycle module, include “with Multicycle” after the version information.
FCS Express for Flow Cytometry Version 6
(De Novo Software, Los Angeles, CA, USA)
If you used the cell cycle module, include “with Multicycle” after the version information.
Kaluza version 1.3
(Beckman Coulter, Miami, FL, USA)
FCAP Array version 3
(Soft Flow, Hungary)
ImageJ
(NIH, USA)
Photoshop version xxxx
(Adobe Systems Incorporated, San Jose, CA, USA)

References

For copyright reasons where references are not available as a Free Full Text, the links point to PubMed, however, I have copies of all references listed below. Therefore, if you are unable to gain access with your own Institutional logins, please contact me and I can forward them to you privately.

For those of you who use FluoroGold for a viability die, this paper by Barber et al (1999) compares FluoroGold with PI and 7-AAD and validates its use as a viability dye

The paper Interpreting flow cytometry data – a guide for the perplexed outlines some data interpretation methodologies, more specifically, those inherent in Biexponential plots, what they mean, why and when they are better than log plots and how to interpret them (it’s a good read for anyone doing data analysis, regardless of write up or not)

The paper Spectral compensation for flow cytometry – visualisation artefacts, limitations and caveats is an excellent reference for the use and purpose of FMO controls as opposed to isotype controls as well as being a good source of information about the importance of proper compensation in flow cytometry.

The paper MIFlowCyte Standards is an excellent resource for understanding the correct publication format for flow cytometry data. If you wish to prepare some data for publication please don’t hesitate to contact Kate for help. I am well versed in how data needs to be presented and described to meet the MIFlowCyt standard, and have all the resources on hand to help make the write up and presentation in this format as painless as possible.

The paper Assessment of Fluorochromes for Two-Photon Laser Scanning Microscopy of Biofilms describes information specific to excitation and emission responses of fluorochromes in 2-photon studies

CCB Cytometry Facility

detmold.staff@gmail.com