BIO RAD S3e User manual

Flow Cytometry/FACS –Bio-Rad S3e Cell Sorter

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Contents
Brief Description............................................................................................................................ 3
Reference ...................................................................................................................................... 3
Location......................................................................................................................................... 3
Suggestion for “Materials and Methods” ..................................................................................... 4
Suggestion for “Acknowledgements” ........................................................................................... 4
People and Contacts...................................................................................................................... 4
Features and Specifications .......................................................................................................... 5
Lasers/Filter-Sets........................................................................................................................... 6
Additional Equipment ................................................................................................................... 7
System Overview........................................................................................................................... 7
WARNINGS and Advice ................................................................................................................. 8
Available Techniques..................................................................................................................... 9
Set Up............................................................................................................................................ 9
Sample Preparation................................................................................................................... 9
Initial Set Up: Start-up and Fluidics Procedure (≈ 20 minutes)............................................... 10
Initial Set Up: Quality Control (≈ 10 minutes)......................................................................... 12
Final Set Up: Low-Pressure Cleaning....................................................................................... 14
Final Set Up: Shut Down.......................................................................................................... 15
Acquisition................................................................................................................................... 18
Sorting ......................................................................................................................................... 21
Other Guidelines ......................................................................................................................... 27
Swapping Fluidics During Sorting: “Hot Swap” ....................................................................... 27
Auto Compensation Wizard .................................................................................................... 29
Protocols and Workspace ....................................................................................................... 32
Further Protocols, Tutorials and Guidelines ............................................................................... 33

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Brief Description
The S3e cell sorter is equipped with two lasers and four fluorescent detectors, plus
both forward and side scatters. Samples are analyzed using the traditional jet-in-air
technology, and events can be sorted at high speeds while maintaining sensitivity and
high-purity.
The S3e cell sorter is largely efficient and user-friendly by automating the complex
presort setup with a unique technology (ProDrop technology). In detail, this cell sorter
brings forward several automated features, such as Startup/shutdown, Stream-to-laser
alignment (Hands-free alignment), Drop delay calculation, Droplet break-off monitoring
and feedback, Phase and deflection adjustments to sort stream, Daily QC reporting and
trending and Collection volume monitoring to prevent overfilling
Reference
Bio-Rad S3eTM Cell Sorter 145-1006
Location
Room 5.22 (Common Equipment Room), 5º floor, Instituto de Tecnologia Química e
Biológica António Xavier (ITQB NOVA)

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Suggestion for “Materials and Methods”
Flow cytometry data were acquired on a Bio-Rad S3e Cell Sorter and analyzed using
the ProSort software, version 1.6.
Flow cytometry data were acquired on a Bio-Rad S3e Cell Sorter operated by the
ProSort software, version 1.6, and analyzed using the Cytospec software from Purdue
University Cytometry Laboratories.
Bacillus subtilis cells were sorted as alive GFP and dead GFP cells using a Bio-Rad S3e
Cell Sorter operated by the ProSort software, version 1.6.
Suggestion for “Acknowledgements”
This work was partially supported by PPBI - Portuguese Platform of BioImaging (PPBI-
POCI-01-0145-FEDER-022122) co-funded by national funds from OE - "Orçamento de
Estado" and by european funds from FEDER - "Fundo Europeu de Desenvolvimento
Regional”.
People and Contacts
Adriano O. Henriques, Microbial Development Group ([email protected])
Mónica Serrano, Microbial Development Group (s[email protected])
Carolina Cassona, Microbial Development Group ([email protected])
Fernando Cruz, Microbial Development Group (fe[email protected])
Phone [phone]
Extension 1524

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Features and Specifications
•Double-Laser System: 488 nm and 561 nm lasers of 100 mW for cell and
particle interrogation
•4 Fluorescence Detectors, photomultiplier tube (PMT) -based
•Forward scatter (FSC) detector, photomultiplier tube (PMT) -based
•Side scatter (SSC) detector, photomultiplier tube (PMT) -based
•Droplet Frequency: 37–43 kHz
•Sorting type: True jet-in-air for high-performance sorting
•Sorting rate: No hardware limitations for sort rate, limited only by droplet
frequency and application
•Sorting purity: >99% pure
•Nozzle Size: 100 µm
•Sorting direction: 2-way sorting
•Sorting collection: Up to 5 x 5 ml tubes each direction; Up to 5 x 1.5 ml tubes
each direction; Microscope slides; 8-well strip each direction
•Sample and collection temperature control system: 4–37°C
•Onboard fluidics and dilution of 8x sheath fluid with deionized water

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Lasers/Filter-Sets
Laser
Filter
Emission
Filter
Fluorophore or
Dye
Fluorophore
Excitation Maximum
Fluorophore
Emission Maximum
488
FL1
BP 525/30
EGFP/GFP
FITC
Alexa Fluor 488
EYFP/YFP
488
495
499
514
507
519
519
527
FL2
BP 586/25
PE
RFP
496, 565
554
578
584
FL3
BP 615/25
PI
PE-Texas Red
536
496, 565
617
619
FL4
655/LP
PE-Cy5
PE-Cy5.5
PE-Cy7
496, 565
490, 565
496, 565
670
693
785
561
FL2
BP 586/25
PE
tdTomato
RFP
DsRed
496, 565
554
554
554
578
581
584
586
FL3
BP 615/25
mCherry
PI
PE-Texas Red
587
536
496, 565
610
617
619
FL4
655/LP
PE-Alexa Fluor 647
PE-Cy5
PE-Cy5.5
PE-Cy7
496, 565
496, 565
496, 565
496, 565
669
670
693
785
BP stands for bandpass filter
LP stands for longpass filter

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Additional Equipment
Computer
•Type: Desktop
•Brand: Dell Inc.
•Model: OptiPlex 3020
•Features: Intel I5 4590 up to 3.7 GHz (quad-core); 4GB RAM; 500 GB HDD
•OS: Windows 7 Professional
•Available Software: ProSort
Monitor
•Brand: Asus
•Model: VE248
System Overview

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WARNINGS and Advice
•Before you get started for the first time on this cell sorter consult the responsible
people for proper training
•For the moment, bacterial pathogens, cell lines or any other agent of Biosafety
level 2 or higher are not allowed, as well as radioactively labelled or flammable
samples
•Do not forget to check the fluidics before you start operating the cell sorter
•Use only MiliQ water in the water container
•While transferring a cap assembly to a new container, avoid touching the
assembly to the outside surfaces of the containers. If it is necessary to lay the cap
assemblies down, set only on pre-sterilized surfaces
•It is recommended that resuspended adherent samples are mixed at high speed
to prevent clumping
•Samples must be filtered prior to running. This will minimize clogs in the sample
tube and nozzle tip
•Do not open the nozzle door
•Keep the green containment door closed as often as possible during the sorting
procedure
•Never try to access the laser beam paths
•Any adjustment to the toolbars should be avoid. Only knowledgeable users must
perform alterations to the current settings. Please, refer to the responsible people for
any constraint
•Optimal settings are automatically determined during the quality control
procedure. Adjusting any of these values will affect the sorting performance
•Do not adjust any setting of the global preferences menu
•Please confirm the bead lot before starting the quality control procedure. If the
bead lot is not in the dropdown list, please refer to the responsible people
•Use always beads from the same lot to perform one quality control procedure
•When the remaining run time is down to 5 minutes, the system will automatically
shut down. The fluidics will need to be refilled and the start-up procedure must be
performed. To prevent this, perform the fluidics “hot swap”.

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•Operate quickly when performing the “hot swap” procedure, as time is limited
to three minutes for the operation. Bring containers of water or sheath fluid in advance.
•The maximum total volumes for a 5 and 1.5 ml collection tubes are 4 and 1 ml,
respectively. Going above these will cause spilling and risk contamination
•You must empty the waste and refill the fluidic containers at the end of the use
•Perform a high-pressure cleaning protocols if you are the last user of the day.
Otherwise, perform a low-pressure cleaning procedure
•Use only 70% ethanol and MiliQ water as cleaning solutions
•Please let us know if some solution or fluidic is running out
•Make sure to save all the data to your own storage device or cloud as we
routinely wipe out the computer’s data
•Do not forget to register your utilization in the logbook. Remember that future
maintenance costs and repairs will be supported by the groups according to the
logbook data
•This equipment is only available for booking in the ITQB intranet
Available Techniques
•Fluorescence-Activated Cell Sorting (FACS)
•Flow Cytometry Analysis
Set Up
Sample Preparation
Cells Suspension
To avoid clogs when working with cells, resuspend cells into a single cell suspension
and always use a 40 µm or smaller filter prior to cell sorting

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Initial Set Up: Start-up and Fluidics Procedure (≈ 20 minutes)
In case you are not the first user of the current day, please confirm with the previous
user whether a low-pressure cleaning final set up has been performed. If so, skip both
start-up and quality control procedures. NEVERTHELESS, DO NOT FORGET TO CHECK THE
FLUIDICS. IF NEEDED, PERFORM THE “HOT SWAP” PROCEDURE (See Swapping Fluidics
During Sorting: “Hot Swap”).
1. Open the bulk fluidics door
2. Detach the quick disconnect from the cap assembly by pushing in the metal
button and lifting the quick disconnect until it engages the magnetic holder above it
3. Slightly lift and pull out the container
4. Uncap the cap assembly from the container

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5. Refill, change or empty the container
6. Carefully place the cap assembly into the container and tighten the cap
7. Place the container into position
8. Attach the quick disconnect to the cap assembly (you should hear a click)
9. Switch on the computer
10. Double-click in the ProSort icon in the desktop
11. Log in into the group account (Contact the responsible people for more log in
options)
12. In the Setup and Maintenance tab, click in the Start-up button
13. Box the Start-up Now option, and then click in the checkmark button of the Start-
up Settings dialog
14. The start-up will be initiated and should take approximately 20 minutes

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Initial Set Up: Quality Control (≈ 10 minutes)
In case you are not the first user of the current day, please confirm with the previous
user whether a low-pressure cleaning final set up has been performed. If so, skip both
start-up and quality control procedures. NEVERTHELESS, DO NOT FORGET TO CHECK THE
FLUIDICS. IF NEEDED, PERFORM THE “HOT SWAP” PROCEDURE (See Swapping Fluidics
During Sorting: “Hot Swap”).
1. Vortex a bottle of ProLine Calibration Beads and place 8-10 drops into a 5 ml
sorter tube. Do not dilute these particles.
2. Place the tube with beads into the sample input station and move the loading
stage into the run position
3. Click in the Run QC button in the Setup and Maintenance tab
4. Choose the correct bead lot in the Bead Lot dropdown list of the Quality Control
Process dialog and then click in the checkmark button to begin the Quality Control
procedure

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5. Once the drop delay calculation has been completed, click in the checkmark
button to accept the drop delay. The QC procedure estimates the optimal drop delay
and performs the side and forward-scatter detectors calibration
6. A QC report window will appear showing the QC test results. If all parameters
have passed the QC test, click in the checkmark button to close the window
7. The system is ready to acquire and sort samples

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Final Set Up: Low-Pressure Cleaning
The low-pressure cleaning procedure should only be performed between uses. If you
are the last user, please perform the shutdown procedure. See Final Set Up: Shut Down
to do so.
1. Fill a 5 ml tube with a minimum of 3 ml of cleaning solution (70% ethanol)
2. Load the tube into the sample station and move it into the run position
3. In the Set Up and Maintenance tab, click in the Clean System button
4. A Clean Warning dialog will open with a prompt warning for the fact that the
tube solution will be rapidly consumed. Click in the Yes option
5. In the Clean System Modes window, select the Low Pressure Clean option and
click in the checkmark button
6. A Clean System process window will be displayed
7. When the cleaning protocol is complete, the prompt “Please put Loading Stage
into the Wash Position” will appear in the Clean System process window. Move the
loading stage into the wash position to close such window
8. Repeat this procedure using MiliQ water as cleaning solution

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9. Empty the waste and refill the fluidics containers using the “Hot Swap”
procedure (See Swapping Fluidics During Sorting: “Hot Swap”)
Final Set Up: Shut Down
The shutdown procedure is only recommended for the end of day’s use. If you are
not the last user, please perform a low-pressure cleaning procedure. See Final Set Up:
Low-Pressure Cleaning to do so.
1. Fill a 5 ml tube with a minimum of 3 ml of cleaning solution (70% ethanol)
2. Load the tube into the sample station and move it to the run position
3. In the Set Up and Maintenance tab, click in the Clean System button
4. A Clean Warning dialog will open with a prompt warning for the fact that the
tube solution will be rapidly consumed. Click in the Yes option
5. In the Clean System Modes window, select the High Pressure Clean option and
click in the checkmark button
6. A Clean Warning dialog will open with a prompt warning for the fact that the
high-pressure cleaning will leave the sorter uncalibrated. Click in the Yes option
7. A Clean System process window will be displayed

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8. When the cleaning protocol is complete, the prompt “Please put Loading Stage
into the Wash Position” will appear in the Clean System process window. Move the
loading stage into the wash position to close such window
9. Click in the Shutdown button in the Setup and Maintenance tab
10. Select the Yes option to confirm that the system should be shut down at this time
11. Select the Yes option to continue without saving protocols. Otherwise, select
Abort, save the protocols and repeat the previous steps.
12. If you intend to use the Automatic Start-up option, box the Yes option and set
both start date and time in the Automatic Start-up window

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13. Yet in this window, select the Yes option for the Run Clean Procedure question
and click in the checkmark button
14. When the prompt “Please Load Cleaning Solution” appears in the Clean and
Shutdown window, load a tube of water MiliQ and move the loading stage into the run
position
15. After the cleaning procedure move the loading stage into the wash position
16. At this time the system is fully shut down. The software will return to the opening
log in screen
17. Close the ProSort software and switch off the computer
18. Empty the waste and refill the fluidics containers

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Acquisition
1. Go to File -> New Protocol or Open Protocol (see Protocols and Workspace)
2. Create at least one histogram and dot plot by clicking in the Histogram and
Density buttons of the Home tab
3. Load the sample into the sample input station and move the loading stage into
the run position
4. Detectors can be renamed for each parameter based on the fluorophores or
markers used
5. Box the Cycle Mode option to allow refresh of the data displayed

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6. Define the target event rate, trigger parameter and threshold value
7. In the Instrument Control Panel window, click in the Start button to begin
sample acquisition
8. Adjust photomultiplier tube (PTM) voltages to place populations
9. Adjust the target event rate, trigger parameter and threshold value
EVENT RATE

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10. Stop sample acquisition by clicking the Stop button
11. Box and define the event limit. Gate Limit can be chosen, and Cycle Mode can
be unboxed at this time. You can enable the Auto Save option to automatically save the
file after the event limit is reached
Cycle Mode can be enabled, or the refresh button can be used to refresh the data as
adjustments are being made
12. Re-collect the sample and save the FCS format file if Auto Save has not been
boxed
13. Unlock the loading stage and remove the sample tube
14. Move the loading stage into the wash position before running the next sample
When running multicolour experiments, compensation is necessary. To use the Auto
Compensation wizard, single stained compensation control samples must be saved in
addition to the multi-coloured samples