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S3 Cell Sorter Instruction Manual

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6.4 Sort Plots

During a sorting experiment a sort plot can be created in the current protocol. This plot  
displays graphically the counts of events (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 designations as assigned in the Sort Logic window 
(Figure 74). Regions can be applied to the sort plot. These regions can be applied to other 
histograms or density plots.

Fig. 77. Sort Plot window.

Содержание 145-1001

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

Страница 2: ......

Страница 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...

Страница 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...

Страница 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...

Страница 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...

Страница 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...

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

Страница 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...

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

Страница 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...

Страница 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...

Страница 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...

Страница 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...

Страница 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...

Страница 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...

Страница 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...

Страница 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...

Страница 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...

Страница 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...

Страница 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...

Страница 22: ...12 S3 Cell Sorter Instruction Manual 12...

Страница 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...

Страница 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...

Страница 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...

Страница 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...

Страница 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...

Страница 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...

Страница 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...

Страница 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...

Страница 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...

Страница 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...

Страница 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...

Страница 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...

Страница 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...

Страница 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...

Страница 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...

Страница 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...

Страница 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...

Страница 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...

Страница 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...

Страница 42: ...S3 Cell Sorter Instruction Manual 32...

Страница 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...

Страница 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...

Страница 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...

Страница 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...

Страница 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...

Страница 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...

Страница 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...

Страница 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...

Страница 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...

Страница 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...

Страница 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...

Страница 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...

Страница 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...

Страница 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...

Страница 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...

Страница 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...

Страница 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...

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

Страница 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...

Страница 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...

Страница 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...

Страница 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...

Страница 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...

Страница 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...

Страница 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...

Страница 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...

Страница 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...

Страница 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...

Страница 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...

Страница 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...

Страница 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...

Страница 74: ...S3 Cell Sorter Instruction Manual 64...

Страница 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...

Страница 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...

Страница 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...

Страница 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...

Страница 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...

Страница 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...

Страница 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...

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

Страница 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...

Страница 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...

Страница 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...

Страница 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...

Страница 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...

Страница 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...

Страница 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...

Страница 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...

Страница 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...

Страница 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...

Страница 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...

Страница 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...

Страница 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...

Страница 96: ...86 S3 Cell Sorter Instruction Manual 86...

Страница 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...

Страница 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...

Страница 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...

Страница 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...

Страница 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...

Страница 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...

Страница 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...

Страница 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...

Страница 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...

Страница 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...

Страница 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...

Страница 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...

Страница 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...

Страница 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...

Страница 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...

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

Страница 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...

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

Страница 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...

Страница 116: ...106 S3 Cell Sorter Instruction Manual 106...

Страница 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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Страница 120: ...65 Germany 089 31 884 0 Greece 30 210 9532 220 Hong Kong 852 2789 3300 Hungary 36 1 459 6100 India 91124 4029300 Israel 03 963 6050 Italy 39 02 216091 Japan 81 3 6361 7000 Korea 82 2 3473 4460 Mexico...

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