In this issue

SpectroFlo New Features ......... 2

Defect Fixes .......... 3

Known Defects ...... 3

Aurora Users Community ........ 3

CytekAssaySetting .................... 4

Video Tutorials ......................... 4

Coming Soon ............................ 4

Troubleshooting Data .............. 5

Michele Medical Leave ........... 5

Aurora Software update

The Cytek Aurora has been updated to the latest release of SpectroFlo v2.2.0. A description of the changes in this version starts on page 2. The Cytek team is continually working to improve the software function-ality as well as the user interface and they welcome suggestion and feed-back on issues.

Cytek & De Novo Live Webinar

Learn more about the latest version of SpectroFlo and FCS Express 7 that can help you optimize your multicolor panels

In the first half of the webinar, you will learn how to use the Similarity

Index in Cytek's SpectroFlo v2.2 software release to QC your reference

controls, and how to use the Complexity Index to compare different iter-

ations of your multicolor panel. In the second half, learn about new tools

and updates to FCS Express 7 that help support spectral data sets includ-

ing spectral plots, new NxN plot functionality, multi-parameter overlays,

and other novel visualization to help you move from raw data to final

results quickly and efficiently.

Presented By:

Janelle Shook- Cytek Biosciences

Sean Burke - De Novo Software

Date: Tuesday December 3rd 3 PM ET / 12 PM PT

Please pre-register here

November 2019 Volume 1, Issue 1

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OVERVIEW SpectroFlo version 2.2.0 reduces throughput time for plate acquisition and extends gating capabili-

ties in the worksheet by adding logical AND and OR gates along with staged quad gates. It improves the workflow and usability of create/update reference controls. Some existing software defects were resolved, and some new software features have also been implemented. All is described in detail below.

SpectroFlo 2.2.0 Release notes

NEW & IMPROVED FEATURES

• Logical AND and OR gates: You can create a Boolean AND or OR gate by multi-selecting two or more gates from the Population Hierarchy,

right clicking on the selection, and selecting “Intersect (And) Gates” or “Join (Or) Gates”.

• Staged Quad Gate: The quad gate feature has been extended such that either the horizontal or vertical split line can be broken and adjusted

left/right or up/down.

• Preference for Skipping Boost: To minimize sample loss when starting acquisition, a preference option has been added allowing you to skip

the initial boost of sample up to the flow cell (referred to as “BOOST_RUN”) for medium and high flow rates.

• Protected CytekAssaySetting: CytekAssaySetting cannot be overwritten by either administrator or operator users. When changes are made

to CytekAssaySetting, you are forced to “Save As” if you want to keep the changes.

• Append FCS Data When Creating Reference Controls in the Library: If a library reference control is recorded with an insufficient number of

events, users can now append FCS data onto the previous acquisition instead of being forced to overwrite previously recorded FCS data.

• Improve Usability in Create Reference Controls: Users now have the flexibility to access the cytometer menu operations, such as SIT Flush

and Clean Flow Cell, and can modify and save user settings within the Create Reference Controls workflow.

• Preference for Bubble/Clog Detection: A new option has been added in the Preferences Acquisition to enable bubble/clog detection.

• Linkage of Worksheet to Experiment: When you right-click on an experiment in the My Experiments list and select Export, the exported ZIP

file now includes linked worksheets and maintains the link between a tube or well at its linked worksheet. Users no longer need to export/

import worksheets associated with the experiment from the Library module and manually link them.

• Allow Users to Import System Defined Fluorescent Tag Groups: In previous software versions, users are not allowed to import system de-

fined fluorescent tag groups. Users can now import a system defined tag group. All user added fluorescent tags will be imported and merged to

the existing tag group in the system.

• Improved Keywords Usability: Users can copy and paste keywords across tubes and wells in the Create New Experiment wizard. In addition,

users can specify which custom keyword can be automatically updated according to the specified value in library.

• Improved Unmixing Accuracy When Reusing Reference Controls from Duplicated Experiment: Each reference control created in an experi-

ment is now normalized based on the Daily QC that was run immediately before the control was acquired. Therefore, reference controls in an

experiment acquired days or weeks before a sample tube is acquired can be used to unmix the tube and the same accurate results can be ob-

tained as unmixing with controls from the library. Now, you can safely duplicate an old experiment to reuse reference controls contained in the

experiment without concerns about instrument drifting causing unmixing inaccuracy.

• New Fluidics Shutdown & Clean Flow Cell Procedure: To improve the maintenance of the flow cell and sample line, the Fluidics Shutdown

and Clean Flow Cell procedures have changed. The Fluidics Shutdown procedure now incorporates 4 tubes (previously 2 tubes) and takes ap-

proximately 7 minutes to complete. We recommend running a Fluidics Shutdown after each experiment is acquired to help keep the flow cell

clean for the next user. The sequence of tubes is as follows: first tube with DI water, second with 10% bleach, third with DI water, and fourth

with 30% contrad.

The revised Clean Flow Cell procedure now incorporates 2 tubes (previously 1 tube), where the first tube contains 10% bleach and the second

tube contains DI water.

Aurora User Community

If you would like to join the

Cytek Aurora User community

reach out to Julie Hill

jhill@cytek.com

Users can ask questions and

give feedback directly to Cytek

application scientists and engi-

neers improving the communi-

cation of new features, bug

fixes and innovative ideas.

Once you have emailed Julie

you will receive an invitation to

create an account by going to

Cytekbio.com/pages/aurora

DEFECT FIXES IN v2.2.0 RELEASE

• Link to FCS File Lost when Renaming to Switch 2 Tubes’ Names: When a user renamed

tubes and groups in a specific way, such as switching existing recorded tube names, the

link between the tube and the FCS file was lost so the UI made it look like the FCS file was

gone. This defect has been fixed.

• Close Experiment to Release FCS Files: In some specific scenarios even after an experi-

ment was closed, some FCS files were not released by the software. Therefore, if you

went to the CytekExport folder and tried to copy the FCS file, Windows system would

display an error. This issue is corrected.

• SpectroFlo Cannot Open Due to Corrupt PathSettings: In a very rare scenario, Spectro-

Flo cannot open due to a corrupted PathSetting saved by the software. This defect has

been fixed.

KNOWN SOFTWARE DEFECTS:

• Post-Acquisition Unmixing File Size Constraint: We recommend only unmixing multi-

color FCS files that contain fewer than 10,000,000 total events. Files that are bigger than

10,000,000 events may take several hours to unmix.

• Events to Display & Live Unmixing: If you want to display a lot of events (e.g.

1,000,000) while live unmixing a tube, the software will become very slow. We recom-

mend reducing your events to display to 50,000 events or less when live unmixing your

data. After the acquisition is finished, you can display 5-100% of your recorded events

without being penalized.

• Auto-Scaling of Axes for Unmixed Files: Auto-scaling of plot axes (found in Plot Proper-

ties) does not always apply properly for an unmixed tube. You may need to toggle to Man-

ual Scale and then back to Auto Axis Scale to get the scaling to update and apply correctly.

• Limitation for the Name of User Defined Keyword: “Name” cannot be used as the key of

a user defined keyword. Software does not prevent users from doing so. However, if users

do define such a keyword and this keyword is added in the statistics table, batch analysis

cannot be completed due to a conflict with a reserved string used in the statistics table of

SpectroFlo.

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Known bug:

Auto-scaling doesn't update on plots, users

need to toggle to manual and back Auto Scale

when creating new plots or changing the axis.

SpectroFlo 2.2.0 Release notes

CytekAssaySettings

The new version of the Spec-

troFlo software no longer al-

lows users to overwrite the

original CytekAssaySettings if a

user makes changes to the

settings, even just adjusting

the FSC and SSC will require

the user to “save as” the new

settings. Renaming to the cell

type or experiment specific

details to recall later will make

it easier to keep track of multi-

ple settings files. If the user

does not “save as” while the

experiment is open when the

experiment is closed the soft-

ware will ask to save changes if

the user clicks Yes to save the

new settings will be named

“CytekAsssaySettings—Copy n”

Cytek and BioLedgend are teaming up to provide the Cell Analysis Fa-

cility Users a workshop for better understanding controls for Spectral

analysis. Look for more information in the coming weeks.

Tentative Date January 22nd

Cytek Video Tutorials

Cytek has complied a wide variety of

video tutorials that are available

online.

New feature in SpectroFlo v2.2 for

re-using reference controls allows

running them from the reference

Library or from inside an experi-

ment both will be updated based on

the daily QC of the instrument.

Here is a video tutorial for creating

the references controls .

A new feature that is included for

reference controls is Benchmarking

User can assign a control as a bench-

mark for comparing to in subsequent

experiments. This could be very use-

ful for tracking how tandem dyes are

performing and whether or not it is

time to replace them .

For more tutorials visit

https://cytekbio.com/blogs/

resources/tagged/video-tutorials

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LEARN MORE

Coming Soon

Michele will be on Medical leave December 3rd – January 2nd. Reponses to email will be very limited during this time.

The facility will try to maintain assisted service during my absence but there may be times that only self-users will be permitted to use the facili-ty. During my absence, please make sure to communicate directly with Kyle if you need assistance for setting up and running experiments. The scheduling calendar includes a Staff schedule for updates, it is located in the top row of the calendar and has up to date information on staff avail-ability.

Scheduling Calendar

For flow cytometry related questions or scheduling, please contact Kyle Herstad 543-7006, email herstky@uw.edu or (lab email) flow-lab@uw.edu

For billing questions, please contact David Cameron camerond@uw.edu

For Administrative questions, please contact Wendy Star wkstar@uw.edu

Michele out on medical leave

Troubleshooting Data

Recently, a user was struggling

with analyzing some data where a

population of interest was not

well resolved and the expected

downstream population con-

tained cells that biologically

should not be present.

The usual gating strategy of de-

fining the double positive CD11b

and Ly6G as Neutrophils was re-

sulting in a 25.7% population of

Microglia (TMEM119 + Ly6C - )

Due to the inability to clearly re-

solve the double positive neutro-

phil population. The spectral spill-

over from PE-Cy7 was impacting

the relatively dim Ly6G in BV711.

Since this is a critical part of the

analysis something needed to be

done to get data that was going to

be useful for the subsequent

study.

One option was to redesign the

panel to better separate the

markers and avoid the spreading

error. This would require a lot of

time, money and loss of valuable

data already collected.

The other option, find a new

gating strategy that resolved the

neutrophils. Plotting Ly6G vs Ly6C

was the answer the dim Ly6G

population was more easily re-

solved and the microglia were not

spilling into the subset. Problem

solved no redesign and the al-

ready collected data was now

easily analyzed.

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