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S3 Cell Sorter Instruction Manual

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This chapter describes the hardware of the S3 system. Understanding the system’s hardware is
essential for proper operation.

2.1 System Overview

The S3 system consists of fluidics, optics, electronics, and software. It can be broken down
into several subsystems (Figure 1).

Hardware Description

Access to nozzle assembly

Access to internal
fluidics system

Access to filters

Touch locking system

Sort collection area

Loading stage

Fig. 1. Front view of the S3 system.

Summary of Contents for 145-1001

Page 1: ...S3 Cell Sorter Instruction Manual Catalog 145 1001 145 1002...

Page 2: ......

Page 3: ...rieval system without permission in writing from Bio Rad Laboratories Bio Rad reserves the right to modify its products and services at any time This instruction manual is subject to change without no...

Page 4: ...ers Maintenance and repairs should be carried out only by authorized service support engineers For service support in the United States and Canada call 1 800 424 6723 toll free and select the technica...

Page 5: ...e due to laser radiation if the associated directions are not followed correctly CAUTION Biohazard This symbol identifies components that may become contaminated with biohazardous material Instrument...

Page 6: ...Electrical Equipment for Measurement Control and Laboratory Use Part 1 General Requirements n UL CSA 61010 1 2012 3rd Ed Standard for Safety Electrical Equipment for Electrical Safety USA Canda NRTL n...

Page 7: ...s The S3 system includes a waste container that may potentially contain hazardous chemical materials depending on the sample used Explosive or Flammability Hazards The S3 system poses no uncommon haza...

Page 8: ...S3 Cell Sorter Instruction Manual vi vi...

Page 9: ...ters 62 Chapter 6 Sorting 65 6 1 Sort Setup 65 6 2 Sort Modes 69 6 3 Sort Statistics 69 6 4 Sort Plots 71 Chapter 7 Additional Software Features 73 7 1 Debubble 73 7 2 Unclog 73 7 3 Swap Tip 73 7 4 Cl...

Page 10: ...S3 Cell Sorter Instruction Manual viii...

Page 11: ...er deflection of the correct droplets proper instrument setup is integral to achieving high purity sort results By automating the complex presort setup with a unique technology the S3 cell sorter crea...

Page 12: ...Plastic box 1 each n Computer CPU and 24 inch monitor 1 each catalog 145 1066 n ProFlow 8x Sheath Fluid preservative free 5 x 4 L 1 case catalog 145 1082 n ProLine Calibration Beads 3 x 5 ml 1 pack ca...

Page 13: ...er operation 2 1 System Overview The S3 system consists of fluidics optics electronics and software It can be broken down into several subsystems Figure 1 Hardware Description 2 Access to nozzle assem...

Page 14: ...ort to connect the system to the computer for communication Ethernet port green use this port to connect the system to the computer for communication There is a color coded guide for proper connection...

Page 15: ...fluid If preferred 1x sheath fluid may be used instead In this case the DI water container will not be used to dilute the sheath but only for rinsing and cleaning If 1x sheath fluid is used ensure the...

Page 16: ...t required When a sample is loaded and ready to run the loading stage can be moved into the run position To move the loading stage into the run position 1 Push the handle on the loading stage down 2 P...

Page 17: ...tage into the run or wash position by holding onto the handle for a smooth movement into position Note If the loading stage is in the wash position and another wash is required press the touch locking...

Page 18: ...k hazard The nozzle door is interlocked and will disable stream charge when opened Only qualified and trained personnel can override this interlock 2 2 4 Sort Collection Chamber The deflection plates...

Page 19: ...regulations to ensure proper handling and disposal of biohazardous substances including samples sorted fractions and waste CAUTION Shock hazard The sort deflection plates in the sort chamber are char...

Page 20: ...ld be collected by changing the filter 2 3 6 Optical Filters Optical filters are coated pieces of glass used to divide the spectrum of light into bands for analysis By separating and detecting differe...

Page 21: ...erlocks have been disengaged Note When opened the nozzle door will close the laser shutters to prevent laser light exposure This interlock will also turn off the drop drive and the stream IMPORTANT Op...

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Page 23: ...y be added and will be logged with the user session in the user report for administrative use These notes will appear in the user report when viewed Note If an automatic startup has been scheduled the...

Page 24: ...reports Print user reports Change user password Change user rights Delete users Create users Edit users Reset other user passwords 3 1 Main Software Window The main software window contains tools for...

Page 25: ...also be selected at this time The protocol used to acquire the data will be loaded from the FCS file Load instrument Opens an existing protocol s settings and loads them into the current protocol sett...

Page 26: ...rotocol The histogram can be resized by dragging the bottom right corner Density Adds a new density plot to the current protocol The density plot can be resized by dragging the bottom right corner Sor...

Page 27: ...ted as the collection vessel Individual mode and limits may be set for each 5 ml tube in each direction 1 5 ml tube Indicates that 1 5 ml tube is selected as the collection vessel Individual mode and...

Page 28: ...m if auto startup has not been selected Shutdown Shuts down the system The shutdown button is not active unless the loading stage is in the wash position The shutdown button will prompt to clean syste...

Page 29: ...llows an image to be added to the workspace The image can be moved or deleted If added prior to saving this image will be saved to the FCS file s embedded protocol Basic Adds basic information to the...

Page 30: ...acquisition button Eject sample tube button Auto save sample upon reaching limit Preset sample event rate button Enter sample event rate Event limit option Cycle mode option Gate limit option Instrum...

Page 31: ...locks the sample The loading stage can be moved into wash position to access sample The touchscreen locking system can also be used to unlock the sample Adjust Sample Starts or stops agitation of the...

Page 32: ...nm 561 nm and 640 nm labels Table 21 PMT controls and their functions Button Name Function Editable The name for each parameter is editable and will be saved with the parameter acquired FCS data The e...

Page 33: ...offset sample pressures which returns the preset Pressure Offset buttons low med high to their correct ranges Global preferences Change global instrument options for sheath concentration used idle shu...

Page 34: ...firm drop delay These puddles will sort 100 events at the current drop delay 1 the current drop delay and the current drop delay 1 CAUTION Optimal settings are calculated automatically after performin...

Page 35: ...struction Manual 25 Fig 19 Droplet Controls window with drop settings highlighted Calibrate droplets Recalculate drop delay Verify drop delay Close window Fig 20 Streams Control window with Enable Tes...

Page 36: ...er stream The stream is visualized as a small bright spot that is illuminated by the stream laser 2 Check the Enable Test Pattern check box the system will charge the side streams to simulate a sort F...

Page 37: ...23 Passwords can be reset and users can be notified to reset their passwords from this window To edit a user s information 1 Click the pencil icon next to the user s name 2 Modify user information in...

Page 38: ...nt rate of 0 when running calibration beads in the low setting or if it shows 500 events the offset will need to be reset Using this tool will reset the differential offset sample pressure which will...

Page 39: ...nning the system to avoid diluting the solution Idle Shutdown Time The system has the ability to shut down automatically and start the shutdown procedure after sitting idle for a designated time perio...

Page 40: ...ptions that can be chosen Histogram and Density Plot axis parameters can be defined and set in the global preferences The user can choose the default axis parameter type Area Height or Width In additi...

Page 41: ...mes on the instrument panel Default Save Location The software can autosave after each file has finished acquiring Files can be saved to a default destination folder on the computer This can be set fo...

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Page 43: ...cking Bulk Fluidics Prior to starting the ProSort software open the bulk fluidics door and check the volume of each container Ensure that the waste container is empty and the sheath fluid and DI conta...

Page 44: ...us for safety 4 2 Logging In To use the S3 system the ProSort software is required The software is preinstalled on the computer that came with the instrument 1 Double click the ProSort software icon l...

Page 45: ...ed up at the moment or a scheduled startup can be designated Startup Now is the default option 4 Choose startup option in the Startup Settings window 5 Click the checkmark box to proceed Note If sched...

Page 46: ...ality Control The quality control QC procedure must be run every day to ensure optimal system performance This procedure requires the ProLine calibration beads to adjust alignment verify system agains...

Page 47: ...us passed exit the window The system is now ready for sample acquisition and sorting If the QC status failed rerun the QC procedure up to two more times 4 4 1 Droplet Setup The first step in the QC pr...

Page 48: ...ving the slider bar The Enable Test Pattern button should be selected in order to apply a charge to the stream Users with administrator privileges may access and modify these settings in the Administr...

Page 49: ...ely calculates the drop delay on the S3 system by analyzing the waste stream while running calibration particles Events are detected normally at the nozzle and then again in the waste stream below The...

Page 50: ...during use The changes required for this are completed automatically To verify that the drop delay is being maintained use the Droplet Monitor button on the tool bar This window will note if the syst...

Page 51: ...ministrator tab For more information refer to Section 3 3 Administrator Tab Toolbar CAUTION Adjusting any of these values will affect the overall system performance For each fluorescent parameter two...

Page 52: ...will appear indicating items within the window can be changed 5 Change the parameters resize window add regions bars or quadrants apply gates or show statistics within this window 6 Repeat for other p...

Page 53: ...e settings to be loaded The preexisting settings will be loaded into and displayed in the current protocol and will be ready to use for acquisition or sorting 4 5 3 Saving Protocols Protocols can be s...

Page 54: ...the information contained in each protocol 4 5 5 Opening FCS Files The ProSort software allows an FCS file to open into two different types of protocols Figure 46 The data can be embedded into the cu...

Page 55: ...an be saved after acquisition or sort The protocol used during the acquisition or sort is embedded in the FCS file The file can be opened at a later time for analysis or to be used as a template for a...

Page 56: ...lick the red X in the upper right corner This will delete the selected plot Figure 48 4 5 10 Changing Parameters To change the parameters displayed in a plot click the dropdown arrow and select a para...

Page 57: ...istics Changes in the settings will be saved with the protocol Figure 50 Refer to Section 3 3 8 Global Preferences for information on how to change the default statistics To remove statistic headings...

Page 58: ...lations of interest A bar region is the only option on a histogram and all other regions will be unavailable Density plots may have ellipses rectangles quadrant or polygon regions There are two ways t...

Page 59: ...ete right click within the region and choose Remove region 4 5 14 Creating Gates Gates can be used between plots once regions have been created The purpose of gating is to limit the events shown in on...

Page 60: ...50 S3 Cell Sorter Instruction Manual 50...

Page 61: ...or open a protocol Note At least one histogram or dot plot must be created to continue 2 Load the sample onto the sample input position and move the loading stage into the run position 3 Detectors ca...

Page 62: ...tions Refer to Section 3 2 Control Panel for an overview of the instrument control functions 5 1 1 Target Event Rate The event rate is related to the sample concentration Generally for every 1 000 000...

Page 63: ...splayed while changing the settings Figure 55 Cycle mode is set in units of seconds and will progressively display events that fall within that period of time Older data will be not be displayed Use t...

Page 64: ...ntensity level to eliminate any background noise and debris while still allowing real events to be detected and analyzed 5 1 6 PMT Voltages Each parameter is collected by a photo multiplier tube PMT I...

Page 65: ...ing and Washing After the sample is unlocked the loading stage can be moved to the wash position The wash process includes a backflush and an exterior wash of the sample line This process is crucial t...

Page 66: ...e 60 As compensation is adjusted the compensation matrix will be updated At any time the compensation matrix can be viewed using the button on the toolbar Figure 61 Fig 60 Same data plots showing inco...

Page 67: ...FCS files must be saved prior to compensation To use the compensation wizard 1 Run single controls for each parameter and your multi stained sample 2 Save FCS files 3 Select Home Compensation Wizard...

Page 68: ...ion box on the axis of the desired histogram or density plot to display the compensated data and acquire or sort 14 The compensation matrix may be viewed at this point if desired The hyperlog feature...

Page 69: ...in your experiment The process is most accurate if you have both positive and negative events in each sample and it is advisable to acquire at least 5 000 positive events After the sample is acquired...

Page 70: ...imate run time is down to an hour and will continue to alert every 15 min until a swap is performed A hot swap can be done when the limiting fluid is highlighted The alert may be snoozed at this point...

Page 71: ...ly 8 Empty the waste container following lab procedures for liquid biohazardous and chemical waste removal 9 Place the waste cap assembly into a new designated waste container or recap emptied waste c...

Page 72: ...ous for safety 5 4 Optical Filters The optical filters separate and direct the fluorescent light to the PMTs for detection These filters are located behind a sliding panel on the top and toward the re...

Page 73: ...n the dichroic and bandpass filters The optical filters within the block are user changeable and will accommodate multiple filter sizes as described in Table 23 The dichroic filter holders are locked...

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Page 75: ...if desired 4 Place filtered sample into the sample input station 5 Move the loading stage into the run position 6 Click Start on the instrument control panel to acquire a small amount of the sample 7...

Page 76: ...indow can be reopened using the Sort Statistics button on the Home toolbar Click on the arrow in the upper right corner to expand the window and show the statistics table 16 If no limits have been set...

Page 77: ...iod The minimum recommended volume is 0 5 ml of filtered buffer or media for each 5 ml tube The minimum starting volume for 1 5 ml tube is 0 1 ml Note The sort output may be temperature controlled to...

Page 78: ...s 2 Right click in the region to sort and select Right Sort or Left Sort That region will change color and any regions used in the gating of that plot will change as well Regions used in sort criteria...

Page 79: ...ents are present sorting will still occur and recovery are important Enrich This mode will sort all positive events regardless if negative events Sorts when recovery is critical are nearby Purity will...

Page 80: ...cond as a rate Aborts per second The abort count per second as a rate Sort Number of sorted events divided by total events x 100 Abort Number of aborted events divided by total events x 100 Efficiency...

Page 81: ...vents sorts and aborts per tube well slide spot in the sort run The data on this sort plot Figure 77 correspond to the Sort Statistics window Figure 76 The numbers 1 10 are the sort position designati...

Page 82: ...72 S3 Cell Sorter Instruction Manual 72...

Page 83: ...nd Maintenance tab toolbar as a first line action The S3 system has the ability to backflush the nozzle potentially releasing the clog and flushing it into the waste If successful run a couple of debu...

Page 84: ...ocedure To clean the system 1 Fill a 5 ml tube with a minimum of 3 ml of cleaning solution 2 Load tube into the sample station and move in to run position 3 Click the Set Up and Maintenance tab 4 Clic...

Page 85: ...ady Can begin acquisition or sort Acquiring sorting Yellow Information Perform requested action Require QC before proceeding Fluids Low High Orange Warning Nothing system is running Calibrating Red Er...

Page 86: ...on at the bottom of the report This report may also be printed if desired 7 7 Printing Print and print preview buttons are available in the File dropdown menu and on the Setup and Maintenance tab Figu...

Page 87: ...inted for reference and will include the session notes entered when users were logged into the software Fig 86 User report The second QC report is a trending report in which a range of dates can bev v...

Page 88: ...idered to be in Class I containment IMPORTANT If the airflow through the HEPA filter falls below required limits for containment the software will alert the user and stop the sort or acquisition immed...

Page 89: ...for dealing with ethanol or bleach Note At any time the shutdown procedure can be canceled by clicking the X icon on the dialog windows Note If a user chooses to log off from the software the force c...

Page 90: ...to continue or No to abort shutdown procedure The shutdown procedure will display an automatic startup option For ease of use and efficient workflow the system can be automatically started the next d...

Page 91: ...ution 7 Move the loading stage into the run position 8 The system will automatically run through the cleaning procedure which takes approximately 2 min while displaying status windows Figure 92 9 Put...

Page 92: ...entered the countdown will begin IMPORTANT It is advised to clear waste and refill fluidic containers at the end of the day s use For automatic startup waste must be empty and 8x sheath fluid and DI w...

Page 93: ...to avoid spraying or flooding A warning will appear and the system may be manually started to resolve the issue 9 1 Scheduling an Automatic Startup There are three ways to schedule an automatic start...

Page 94: ...he system a countdown will appear at the bottom of the login window Figure 97 This countdown indicates how much time is left before the startup procedure will begin Note The software must be opened to...

Page 95: ...ly scheduled automatic startup may influence other users plans Be sure to check with others when choosing this option Table 28 Auto startup buttons Button Command Function Cancel Cancels the previousl...

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Page 97: ...ll the fluidics at the end of use to ensure no errors will occur 10 1 2 Weekly Each week wipe down the system with a mild disinfectant Clean any salt crystal buildup in the sort chamber or on the defl...

Page 98: ...e could help free the clog This is run through the sample input station Cleaning solution can dislodge or loosen clogs to free up the line or the nozzle tip If successful run at least two rinse cycles...

Page 99: ...ree cloth in the nozzle chamber under the nozzle tip to prevent dropping the nozzle into the instrument 8 Rotate the black collar holding in the nozzle until it comes loose Note There is a small O rin...

Page 100: ...peat if necessary until the stream is coming out straight 13 Proceed to step 14 14 Place the O ring at the point on the nozzle where the bottom of the black collar will be 15 Place the nozzle tip back...

Page 101: ...the sort collection area 21 If not coarse adjustments can be made by physically moving the black block area at the top of the nozzle Note The upper right gimbal moves the nozzle right to left The bott...

Page 102: ...t on the stream by scanning forward and back or side to side 23 Adjust the gimbals with the hex wrench as they are highlighted by the wizard 24 Count turns and visually estimate where the stream looks...

Page 103: ...ent process should complete the alignment The window will give an option of repeating the gimball adjustment process or skipping and proceeding to droplet calibration If the camera views show that the...

Page 104: ...removed Replace filter into the S3 system 10 6 Disinfectants Always follow the PPE guidelines relevant to your laboratory s safety procedures for dealing with the disinfectants recommended below Disi...

Page 105: ...ing basic cellular sterile technique The bulk fluidics can also contribute to contamination despite having internal filters built into the lines To run the Decontaminate tool 1 Move the loading stage...

Page 106: ...r and replace 10 Click the checkmark button to proceed The system will drain the internal sheath reservoirs into the waste container Figure 113 The system will directly prime the fluidics with the dec...

Page 107: ...fore priming the system with 8x sheath fluid or DI water Figures 118 119 After decontamination the system should be ready for running samples Any high background should be eliminated In order to decon...

Page 108: ...osen The system will soak and go through an extended warmup Figure 120 before a system startup is commenced Figure 121 After decontamination the system should be ready for running samples Any high bac...

Page 109: ...Sample is currently running Stop sort or acquisition QC will not pass Calibration beads diluted Use the undiluted ProLine beads directly from bottle Using wrong calibration beads Use only ProLine bea...

Page 110: ...cover so it is seated proper position after nozzle been moved during tip swap correctly Once in the proper place the tip swap nozzle stage can be lowered into position Cannot acquire sample Room too...

Page 111: ...tact Bio Rad Technical Support 40 000 eps Threshold may be set too low raise threshold value Signal intensity is too bright Add ND filter Poor drop delay uneven V Bad side streams or center Set defann...

Page 112: ...102 S3 Cell Sorter Instruction Manual 102...

Page 113: ...Hoboken John Wiley Sons Cytometry Part A Journal of the International Society for the Advancement of Cytometry Wiley http onlinelibrary wiley com journal 10 1002 28ISSN 291552 4930 Purdue University...

Page 114: ...S3 Cell Sorter Instruction Manual 104...

Page 115: ...to stream to laser alignment Sorting Directions Two way Modes Single purity enrich Collection Up to 5 x 5 ml or 1 5 ml sample tube each direction Microscope slides 2 x 8 micro well strip each directio...

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Page 117: ...86 ProLine Universal Calibration Beads 3 x 5 ml Accessory Parts 145 1065 S3 Accessory Kit 145 1084 S3 Empty Fluidic Containers 3 x 4 L 144 1001 S3 Sheath Cap Assembly white 144 1002 S3 DI Water Cap As...

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