S3™ and S3e™ Cell Sorters Instruction Manual Catalog #145-1001 #145-1002 #145-1005 #145-1006 #145-1008
Bio-Rad Technical Support For help and technical advice, please contact the Bio-Rad Technical Support department. In the United States, the Technical Support department is open Monday–Friday, 5:00 AM–5:00 PM, Pacific time. http://www.bio-rad.
Bio-Rad Laboratories Resources Bio-Rad provides many resources for scientists. Table 1 lists available resources and how to locate what you need. Table 1. Bio-Rad resources. Resource How to Contact Local Bio-Rad Laboratories Find local information and contacts on the Bio-Rad Laboratories website by selecting your country representatives on the homepage (www.bio-rad.com).
Safety and Regulatory Compliance For safe operation of the S3 and S3e Cell Sorter systems, we strongly recommend that you follow the safety specifications listed in this section and throughout the manual. Safety Warning Labels Warning labels posted on the instrument and in this manual warn you about sources of injury or harm. Refer to Table 3 to review the meaning of each safety warning label. Table 3. Meaning of safety warning labels.
Safe Use Specifications and Compliance Laser Product Hazard Classification The intent of the laser hazard classification is to identify hazards to users posed by the laser, and provide appropriate protective measures. The S3 or S3e laser is a Class 1 laser product that complies with 21 CFR 1040.10 and 1040.11, except for deviations pursuant to Laser Notice No.
n Wash your hands thoroughly with soap and water after working with any potentially infectious material before leaving the laboratory n Remove wristwatches and jewelry before working at the bench n Store all infectious or potentially infectious material in unbreakable leak-proof containers n Before leaving the laboratory, remove protective clothing n n n n n Do not use a gloved hand to write, answer the telephone, turn on a light switch, or touch anything that other people may touch without glo
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Table of Contents Chapter 1: Introduction 1 Chapter 7: Additional Software Features 73 1.1 System Components 1.2 Installation Requirements 2 2 Chapter 2: Hardware Description 3 7.1 Debubble 7.2 Unclog 7.3 Swap Tip 7.4 Clean System 7.5 Instrument Status Box 7.6 Status Bar 7.7 Printing 7.8 Quality Control Reports 7.9 User Reports 7.10 Biosafety System 73 73 73 74 75 75 76 76 77 78 2.1 System Overview 2.2 Fluidics System 2.3 Optics 2.4 Electronics 3 5 9 11 Chapter 3: ProSort™ Software 13 3.
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1 Introduction The S3™ and S3e™ Cell Sorters are designed to offer affordable, dependable, and simplified cell sorting. As benchtop cell sorters, the S3 and S3e Cell Sorters are equipped with one or two lasers and up to four fluorescent detectors, plus 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.
Introduction 1.
2 Hardware Description This chapter describes the hardware of the S3™ and S3e™ systems. Understanding the system’s hardware is essential for proper operation. 2.1 System Overview The S3 and S3e Systems consist of fluidics, optics, electronics, and software. These can be broken down into several subsystems (Figure 1). Access to nozzle assembly Access to filters Touch locking system Sort collection area Access to internal fluidics system Loading stage Fig. 1. Front view of the S3 system.
Hardware Description 2.1.1 Instrument Back Panel The rear connector panel of the S3 or S3e system includes these features: power switch (black) — press the main power switch to turn on power to the system. WARNING! The main power switch should not be used to shut down the system. Perform system shutdown from the ProSort™ software. For more information, refer to Chapter 8, Shutdown ■■ Main ■■ Power input (black) — plug in the power cord here.
2.2 Fluidics System 2.2 Fluidics System The S3 and S3e fluidics system consists of the bulk fluidics, loading stage, nozzle, and sort collection chamber. The fluidics system supplies sheath fluid, DI water, and sample to the nozzle, and then collects the waste for proper disposal. CAUTION: Biohazard! Biosafety is of utmost importance while operating this instrument.
Hardware Description The sheath fluid and DI water are both filtered through a 0.2 µm filter to remove any particulates from the fluid before circulating through the system. Fluidic levels are monitored by the software using a weight measurement system. Below each fluidic container is a bulk fluidics tray. Each tray contains a sensor that translates volume weight into fluidic run time and helps to keep the system dry while swapping fluidics. IMPORTANT! Replace filter cartridges on a regular basis.
2.2 Fluidics System During washing, a new sample can be loaded into the sample input station. The status of the loading stage is displayed by a locked or unlocked padlock symbol located on the touch locking system screen (Figure 7). Fig. 7. Touch locking system screen. To move the loading stage from the run position to the wash position: 1. Press the touch locking system screen until the padlock displays as unlocked. This will depressurize and unlock the sample station. 2.
Hardware Description 2.2.3 Nozzle The nozzle controls many crucial aspects associated with sorting such as: ■■ Creating a stable vibration to generate droplets ■■ Hydrodynamically focusing the sample ■■ Removing air from the nozzle (debubbling) The nozzle compartment (Figure 8) can be accessed through the top, front sliding door. The nozzle tip is a 100 µm orifice for centering the sheath stream to the laser interrogation point.
2.3 Optics Deflection plates Fig. 9. Sort collection chamber and deflection plates. The sort positions for each collection vessel are numbered and will correlate to the position numbering in the software when the sort logic and limits are set. When placing tubes in the sort chamber for sorting, it is recommended to add media or buffer to the tube to help prevent sorted cells from drying out and to cushion their collection. The minimum recommended volume is 0.5 ml of media or buffer for each 5 ml tube.
Hardware Description 2.3.2 Beam Shaping Optic (BSO) The beam shaping optic sits in between the laser(s) and the interrogation point. This serves to shape and focus the laser beam(s) to optimize the illumination of the cell. 2.3.3 Interrogation The interrogation point is the point at which the laser beam(s) intercepts the core stream of the sample. At this point, light is scattered around each individual particle and the particle fluoresces if any fluorophores are attached. 2.3.
2.4 Electronics 2.4 Electronics The S3 and S3e electronics process and deliver the emitted light signals to the software for user analysis. Also included in this subsystem are the deflection plates in the sort chamber and the safety interlocks. CAUTION! Shock hazard! Only qualified, trained technicians should carry out service work on electronic components due to potential shock hazard. 2.4.
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3 ProSort™ Software This chapter describes the features of the ProSort Software. The software is the main interface for the S3™ or S3e™ Cell Sorter System, giving overall status and providing control. To start the software, double click the ProSort icon (Figure 11) on the desktop. Fig. 11. ProSort Software icon. The login window will appear (Figure 12). This window will also appear when switching users. Enter your user name and password to log in.
ProSort Software Table 7. Control differences between administration and user modes.
3.1 Main Software Window 3.1.1 Title Bar Buttons The title bar of the main software window provides the items listed in Table 8. Table 8. Title bar buttons in the main software window. Button Command Function Save Saves FCS file after acquiring sample Sign out/ Signs out the current user and returns the software to the login window. If Switch user the shutdown procedure was not performed prior to sign out, the system will maintain startup and QC.
ProSort Software 3.1.3 Home Tab Toolbar The Home tab toolbar consist of six sections for basic workspace functions and sort options (Figure 14): ■■ ■■ ■■ ■■ ■■ ■■ Plots — available plots for viewing data. Table 10 describes the plots and their functions Regions — different shapes for selecting region of interest. Table 11 describes the regions and their functions Compensation — perform fluorescence compensation.
3.1 Main Software Window Table 11. Region buttons and their functions (continued). Button Name Function Quadrant Adds a quadrant region to the selected density plot. If a density plot is not selected, this region will be grayed out. The region can be moved, resized, and/or deleted Bar Adds a bar region to the selected histogram. If a histogram is not selected, this region will be grayed out. The region can be moved, resized, and/or deleted Table 12. Compensation buttons and their functions.
ProSort Software 3.1.4 Setup and Maintenance Tab Toolbar The Setup and Maintenance tab toolbar consists of five sections (Figure 15): ■■ ■■ ystem — startup or shutdown the instrument from the software. Table 15 describes the S system buttons and their functions uality Control (QC) — perform or review QC runs. Table 16 describes the QC buttons and Q their functions ■■ Fluidics — access common fluidic functions. Table 17 describes the buttons and their functions ■■ Other — additional features.
3.1 Main Software Window Table 17. Fluidics buttons and their functions. Button Name Function Debubble Removes bubbles from nozzle Unclog Allows user to pull vacuum in the case of a nozzle clog. Use this option before removing the tip for sonication or replacing with a new nozzle tip Swap Tip Stops the sheath flow to allow nozzle tip replacement. A wizard will appear to walk the user through a nozzle change.
ProSort Software 3.2 Control Panel System operation is performed and monitored with the control panel (Figure 16). The control panel consists of three parts: ■■ Instrument control — controls for performing a run ■■ PMT control — controls for modifying the PMTs ■■ Instrument status box — displays the status of your instrument Instrument control buttons are described in Table 20 and PMT control buttons are described in Table 21.
3.2 Control Panel Table 20. Instrument controls and their functions. Button Name Function Start Sample Acquisition Starts sample acquisition. If a data file is loaded and it has been saved, the data will clear and acquisition will start Stop Sample Acquisition Stops acquisition and sample flow. Regions, gates, and annotations may be modified before saving the FCS file Start Sorting Starts sorting.
ProSort Software Table 20. Instrument controls and their functions (continued). Button Name Function Fluidics System Indicates the status of the fluidics system and run time information. When Status the fluidics system is down to an hour of run time, the fluidics status window will automatically open to show which container needs attention.
3.3 Administrator Tab Toolbar Table 22. Administrator tab toolbar buttons and their functions. Button Name Function Edit QC Criteria View and edit the CV and PMT voltage criteria for the quality control procedure. This may be done as a result of a bead lot change Edit Droplets Edit the settings for droplet creation. These settings include drop drive amplitude, drop drive frequency, charge phase, defanning, and drop delay.
ProSort Software Fig. 18. Edit QC Criteria window with green plus button and rewind arrow highlighted. 3.3.2 Edit Droplets Droplet controls are available to the administrator for troubleshooting purposes. While the system is maintaining, the drop delay, drop drive amplitude, and drop drive frequency cannot be adjusted (Figure 19). If any of these settings is adjusted, use ProLine calibration beads to recalculate the drop delay before running samples.
3.3 Administrator Tab Toolbar Calibrate droplets Recalculate drop delay Verify drop delay Close window Fig. 19. Droplet Controls window with drop settings highlighted. Fig. 20. Streams Control window with Enable Test Pattern highlighted.
ProSort Software To check for stream defanning: 1. Locate the center 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. Fanning of the streams can be seen more easily with the center stream than the two side streams. The test pattern will make the center stream slightly broader. 3. Determine if the center stream is one solid stream.
3.3 Administrator Tab Toolbar 3.3.5 Managing Users The Manage Users button opens the Manage Users window. This window allows an administrator to add, delete, or edit users (Figure 23). Passwords can be reset and users can be notified to reset their passwords from this window. Fig. 23. Manage Users window. To edit a user’s information: 1. Click the pencil icon next to the user’s name. 2. Modify user information in the Edit User window (Figure 24). 3. Click Save.
ProSort Software 3.3.6 Decontaminate An administrator can run the decontaminate procedure if the system shows signs of heavy background, noticeable debris when running water, or contamination. The complete internal fluidic system will be decontaminated. Follow the wizard procedure located in Section 10.7, Decontamination. 3.3.
3.3 Administrator Tab Toolbar 3.3.8 Global Preferences Users with administrator privileges have the ability to change global settings for the instrument. Changes to global preferences will become effective upon accepting the changes. Preferences specific to plots will be displayed when new plots are created. CAUTION! These settings are global and may influence other users’ experiments. Adjusting any of these values will change the optimal settings for the instrument.
ProSort Software Boost Adjustment The default boost duration is optimized to bring the sample into position for interrogation by the laser once acquisition is started. Default values can be restored by selecting Default. If this value is adjusted, test the boost duration with a known sample to optimize the duration before running the unknown samples. Force Cleaning There are two methods to end a user session in the software — log out or shut down.
3.3 Administrator Tab Toolbar Parameter Names Each parameter is defaulted to its channel name (FSC, SSC, FL1, FL2, FL3, and FL4). The default parameter name can be entered by the user. This will be the parameter name for all new protocols created once the preferences have been changed. Note: Parameter names can also be changed on the instrument panel but they will revert to default names when a new protocol is opened. Refer to Section 5.1.6, PMT Voltages, to see how to change names on the instrument panel.
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4 Getting Started After system installation by a Bio-Rad certified service engineer, the S3™ or S3e™ System should not be turned off via the main power switch. The system is safe in standby mode after performing a software shutdown of the system. This will help facilitate quick or automated startup. If the system is shut off via the main power switch, always power the instrument on before starting the ProSort™ Software.
Getting Started Sheath fluid cap assembly Sheath fluid container Quick disconnect Fig. 29. Bulk fluidics with sheath fluid components noted. IMPORTANT! When handling sheath fluid and DI water containers, minimize air exposure to help avoid contamination ■■ ■■ ■■ hile transferring cap assemblies to a new container, avoid touching the W assembly to the outside surfaces of the containers.
4.3 Daily Startup Fig. 31. Automatic startup message in login window. 4.3 Daily Startup On a daily basis, the system requires approximately 20 min of warmup time before running quality control or any samples. To warm up the system: 1. Move the loading stage into the wash position. 2. Click on the Setup and Maintenance tab. 3. Click Startup. Automatically the Startup Settings window will pop up (Figure 32).
Getting Started During the startup process, the fundamental alignment window will also open to initiate cameras, maintain droplets, and align streams to the lasers. The startup process also includes a warm up period (Figure 34). Fig. 34. Startup Process window. After startup completion, both windows will close automatically and buttons in the toolbars will become available to use. Note: Automated startup is an option for this instrument. At shutdown, a startup time can be designated.
4.4 Quality Control Note: If the bead lot is not available in the dropdown menu, refer to Section 3.3.1, Editing QC Criteria. A series of windows will open during the QC procedure to perform alignment and calculate drop delay settings. User interaction is not required for these windows. These steps are described later in this section. 7. After completion, the Drop Delay Alignment Status window will open with the optimal drop delay settings (Figure 36A). A B Fig. 36.
Getting Started Fig. 37. Droplet Monitor window. 4.4.2 Side Streams Following droplet setup, the side streams are adjusted to align with the sort tube positions and to ensure they are not fanning. The streams may be viewed by clicking View Streams on the Home tab. The View Streams window displays a charge phase and defanning slider bar (Figure 38). Users can manually adjust the charge phase and defanning values by moving the slider bar.
4.4 Quality Control 4.4.3 Alignment During the alignment phase, automated micro-motors sweep the fluidic stream through the laser beams to find the point of optimal illumination. This automated process requires the stream and laser(s) to be running. After alignment, the CVs and voltages are compared to the QC criteria. During the QC procedure, a calibration protocol will open and run automatically. Upon completion, the data are saved as an FCS file.
Getting Started Fig. 40. Drop Delay Alignment Status window. Recalculate drop delay Verify manually Accept 4.4.5 Drop Delay Maintenance After drop delay determination, the system will be automatically placed into a droplet monitor mode to maintain the break-off 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 system is maintaining (Figure 41). Fig. 41.
4.4 Quality Control 4.4.6 Recalculate Drop Delay To recalculate the drop delay using ProLine calibration beads, click the Recalculate button (Figures 40 and 41). This button will not align the streams or verify CVs. This button will only calculate the drop delay. During the drop delay calculation in the QC procedure, the same recalculate button on the Drop Delay Alignment Status window will be available in the Drop Monitor window.
Getting Started 4.5 Protocols and Workspace Before acquiring samples, a protocol with at least one histogram or density plot is required to visualize the events. The last used protocol will automatically open with the software. A protocol consists of histograms, gates, regions, trigger, threshold, PMT settings, sort logic, compensation settings, and all other information required for a run. Protocols can be used as a template for future runs. To add a new protocol: 1. Click File. 2.
4.5 Protocols and Workspace Fig. 44. File dropdown menu with New, Open, Load Instrument Settings, and Save Protocol highlighted. 4.5.2 Loading Instrument Settings Protocols can be saved for future use. Each protocol is saved with the following information: plots, regions, gates, trigger, threshold, PMT voltages, sort logic, sort limits, sort modes, compensation settings, and filter configuration. To load a previously saved instrument setting: 1.
Getting Started 4.5.4 Displaying Multiple Protocols Multiple protocols can be opened at the same time by opening an existing protocol or a new protocol from the file menu. When multiple protocols are opened, they will be displayed as tabs along the top of the workspace (Figure 45). Selecting between each tab will update instrument settings with the information contained in each protocol. Fig. 45. Workspace with protocol tab highlighted. 4.5.
4.5 Protocols and Workspace 4.5.6 Saving FCS Files When the AutoSave box is not checked, the FCS file can 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 subsequent acquisition. 4.5.7 Saving Partial FCS Files There may be instances when a large number of events will be acquired during a sort.
Getting Started Plots can be moved within the workspace by grabbing the bar at the top and dragging to the desired position. The remaining plots will shift accordingly. To delete a plot, click the red X in the upper right corner. This will delete the selected plot (Figure 48). Fig. 48. Density plot with resizing, moving, and deleting functions highlighted. 4.5.10 Changing Parameters To change the parameters displayed in a plot, click the dropdown arrow and select a parameter (Figure 49).
4.5 Protocols and Workspace 4.5.11 Viewing Statistics To view the statistics on a plot, highlight the plot and select the Statistics dropdown arrow in the lower left corner (Figure 50). To hide the statistics, click the same button. Fig. 50. Density plot with statistics. 4.5.12 Editing and Reordering Statistics There are several options for displaying statistics. Changes in the settings will be saved with the protocol (Figure 50). Refer to Section 3.3.
Getting Started 1. Right click on the Statistics panel. 2. Click on the statistic name and drag and drop to the new location. 3. Check Apply to all to apply to all plots in the protocol. To alter number of digits after the decimal: 1. Right click in the Statistics panel. 2. Find statistic of interest and change the value in the Precision box. 3. Check Apply to all to apply to all plots in the protocol. 4.5.
4.5 Protocols and Workspace A region can be moved, tilted, resized, and deleted (Figure 52). 1. Click the desired region. 2. To move region, hover till the crosshairs icon appears. Click and drag to new location. 3. To tilt, click the green circle indicator above the region and drag to change the angle. 4. To resize, click the circle or square indicators around the region and drag to the appropriate size. 5. To delete, right click within the region and choose Remove region. Fig. 52.
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5 Acquisition Sample acquisition can be done independent of sorting. As a sample is run through the system, data are gathered and can then be saved in FCS 3.1 format. These files can be loaded and analyzed using third party software compatible with FCS 3.1 files. Often, setup samples — for example, unstained samples or single-color compensation samples — are acquired prior to sorting the actual sample of interest. 5.1 Acquisition Setup Prior to running a sample, a protocol must be loaded or created.
Acquisition Note: Cycle mode can be enabled or the refresh button can be used to refresh the data displayed on the screen as adjustments are being made. When running multicolored experiments, compensation is necessary. In order to use the Auto Compensation wizard, single stained compensation control samples must be saved in addition to the multicolored stained samples. 13. Run single stained compensation control for first parameter. 14.
5.1 Aquisition Setup 5.1.2 Event Limit An event limit may be set on the acquisition (Figure 55). This limit will be based on the total events collected. The gate default is All. When this event limit is reached, the acquisition will stop and the FCS file can be saved. The maximum number of events acquired in a single FCS file is 100,000,000. Note: Large FCS files may take time to save, load, and display. Fig. 55. Event limit and cycle mode. A gate limit can be used to define the event limit.
Acquisition 5.1.5 Threshold The threshold level is set using the trigger parameter with a range of 0.1–100% (Figure 56). The purpose of the threshold is to set an intensity 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). Independent voltage control is available for each PMT.
5.2 Compensation 5.1.8 Pausing and Resuming Acquisition may be paused and resumed while in progress using buttons on the instrument control panel. Pausing will stop the sample flow. During the pause, the time parameter within the data file will continue, and will show a time gap when resumed. If you wish to replace the sample tube during the pause: 1. Press the touch locking system screen above the loading stage until the padlock is displayed as unlocked. 2. Move the loading stage into the wash position.
Acquisition Fig. 59. Density plot showing uncompensated (left) and compensated data (right). The same can be done for the FL2 positive control, though the compensation required is much less (Figure 60). Fig. 60. Same data plots showing incorrectly compensated file (left) and correctly compensated file (right). 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).
5.2 Compensation Fig. 61. Compensation matrix. 5.2.1 Auto Compensation Wizard The compensation wizard assists with establishing the proper compensation matrix coefficient for each parameter. Single controls for each parameter and the associated 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. 4. Select the parameters for compensation (Figure 62).
Acquisition 6. Locate and select the single control FCS file for each parameter. 7. Select whether to use height or area for all parameters. 8. Click the forward blue arrow to open the Automatic Compensation Calculation Wizard window (Figure 63). 9. Adjust the scatter, negative, and positive regions as necessary. Note: The positive and negative population gates must not overlap. Fig. 63. Using the auto compensation wizard to adjust positive and negative gates. 10.
5.2 Compensation Fig. 64. Density plot showing hyperlog, log, and compensation options. 5.2.2 Manual Compensation It is advised to use the auto compensation feature of the software, however it is possible to alter the compensation matrix manually (Figure 65). This process, although historically used, is difficult to set correctly and the process of compensation should only be performed using the wizard unless knowledgeable and statistically correct adjustments can be done by manual compensation.
Acquisition 5.3 Checking or Swapping Fluidics The bulk fluidic containers are located inside the system and can be accessed by the fluidics door on the left front of the instrument. These fluidic containers require changing on a regular basis. As recommended in Chapter 4, Getting Started, fluidic containers should be changed prior to initiating the software or after shutting down the system.
5.3 Checking or Swapping Fluidics To perform a hot swap: 1. On the Setup and Maintenance tab select Swap Fluidics. 2. A countdown timer will automatically start. This is the amount of time left to swap the containers before the system will automatically shut down (Figure 67). WARNING! The system contains a limited amount of reserve fluids to continue running the system during the swap. If the timer reaches zero, the system will automatically shut down.
Acquisition 11. Place the container back into position. 12. Attach the quick disconnect to the cap assembly. An audible click should be heard. 13. Click the play button in the Swap Fluidics window to finish the swapping procedure. IMPORTANT! ■■ When handling sheath fluid and DI water containers, minimizing air exposure will help to avoid contamination ■■ ■■ hile transferring cap assemblies to a new container, avoid touching the W assembly to outside surfaces of the containers.
5.4 Optical Filters In front of each PMT is a holder for a neutral density filter or bandpass filter. Each filter holder contains a screw-in retaining ring that can be removed or replaced with the provided red S3 spanner wrench. 5.4.2 Optical Filter Blocks The S3 and S3e Systems include two preset filter blocks. The filter set depends on the instrument configuration. The optical filter blocks can be changed to accommodate dectection of different fluorophores.
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6 Sorting Sorting cell populations is highly dependent on several factors such as cell type, population frequency in the starting sample, cell concentration, sample preparation, and sort logic definitions. It is very important to start with a single cell suspension with minimal amount of dead cells or debris contamination. 6.1 Sort Setup The majority of sort setup procedure is automatic and is completed during daily QC. When ready to sort, the desired populations must be selected using regions and gates.
Sorting Fig. 70. Sort Logic window. 14. Close the sort chamber door. 15. Click Start Sort on the instrument control panel. The Sort Statistics window will open and sorting will begin (Figure 71). The Sort Statistics window 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, click the same sort button to stop the sort when desired. 17.
6.1 Sort Setup 6.1.1 Sort Collection The S3™ and S3e™ Systems offer four types of sort collection vessel options: ■■ 5 ml tubes ■■ 1.5 ml tubes ■■ 8-well strips ■■ Microscope glass slides Each sort direction has the capability of sorting into 5 ml tubes and 1.5 ml tubes up to a quantity of 5 tubes in each direction. Using the quick-attaching adaptors, sorted cells can be collected into 8-well strips (up to a quantity of 8) or onto a microscope slide (up to a quantity of 20).
Sorting Note: If a sort limit is selected and volume tracking enabled, whichever criterion (limit or full tube) is met first will stop the sort. IMPORTANT! The maximum total volume for a 5 ml collection tube is considered to be 4 ml. The maximum total volume for 1.5 ml tube is 1 ml. Going above these maximum volumes will cause spraying and risk contamination. 6.1.3 Setting Regions to Sort Regions and gates in the histograms and dot plots are used to create sort logic.
6.2 Sort Modes Fig. 74. Sort Logic window with sort priority and sort modes highlighted. 6.2 Sort Modes Table 24 describes the three sort modes available when setting up a sort. Sort modes can be selected for each sort direction. Table 24. Sort mode definitions. Sort Mode Definition When to Use Single Cell This mode requires that one and only one positive event be in the center of the droplet without any negative events nearby.
Sorting Table 25 describes the available sort statistics. Each statistic is sort-stream specific. The Sort Statistics window can be placed in the workspace for printing. Sort statistics are saved within the FCS file. Table 25. Description of sort statistics.
6.3 Sort Statistics 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.
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7 Additional Software Features The ProSort™ software is an acquisition and sorting software package with the additional features described in this chapter. 7.1 Debubble During the startup procedure, a debubble step will occur to clear the system of air. At times, a bubble may enter the nozzle and cause problems with the stream. On the Setup and Maintenance tab select Debubble to rinse the system and attempt to debubble the nozzle. The stream can also be monitored at this time to see any changes. 7.
Additional Software Features 7.4 Clean System In between samples or users, cleaning can be done at low or high pressure. High pressure cleaning will cause the system to stop maintaining droplets and a QC procedure must be run before sorting. If cleaning between samples, the low pressure option is recommended. The fluidics lines will be flushed thoroughly with cleaning solution, similarly to the cleaning protocol used during the shutdown or logout procedure. To clean the system: 1.
7.5 Instrument Status Box Fig. 81. Cleaning protocol final stage. The cleaning protocol will be completed when the window disappears. If a high pressure cleaning has been chosen, run a QC using ProLine™ calibration beads before sorting. 7.5 Instrument Status Box The instrument status box is located at the bottom of the instrument control panel. The notification and the color of the box will change depending on the state of the instrument. Table 26 describes the notifications and colors. Table 26.
Additional Software Features 7.7 Printing Print and print preview buttons are available in the File dropdown menu and on the Setup and Maintenance tab (Figure 83). Use the print preview function to move histograms and density plots in your print layout. Regions can also be modified in this view and will apply to the protocol displayed. Once arranged, click the print button to bring up the print dialog where the printer and paper may be selected.
7.9 User Reports The second QC report is a trending report in which a range of dates can bev viewed for PMT voltages, CVs, and drop drive amplitude and frequency (Figure 85). When viewed, comments can be added and the reports may be printed for reference. Fig. 85. QC trending report. 7.9 User Reports User reports are accessible by administrators and can be used to track system usage over time (Figure 86).
Additional Software Features 7.10 Biosafety System The S3™ Biosafety System is a Class I aerosol containment hood designed to work with the S3 and S3e Cell Sorter, which works in conjunction with ProSort™ Software, providing users and the environment protection from aerosols created during the cell sorting process.
8 Shutdown 8.1 Daily Shutdown The S3™ and S3e™ System should be shut down at the end of the day. This will turn off all droplet formation and charging, lasers, and streams. A flush will be completed with the DI water container to rinse the system. IMPORTANT! Always follow the personal protective equipment (PPE) guidelines relevant to your laboratory’s safety procedures for dealing with ethanol or bleach.
Shutdown 3. Click Yes to confirm shutdown. Note: If there are any unsaved protocols, the software will warn you that unsaved changes will be lost (Figure 89). 4. Click Yes to continue or No to abort shutdown procedure. Fig. 89. Unsaved protocols check. The shutdown procedure will display an automatic startup option. For ease of use and efficient workflow, the system can be automatically started the next day (Figure 90). 5.
8.1 Daily Shutdown A window will open requesting a tube of cleaner to be used to clean the system (Figure 91). 6. Load a tube of cleaning solution. 7. Move the loading stage into the run position. Fig. 91. Start the clean system option by following the prompt to load a tube of cleaner. 8. The system will automatically run through the cleaning procedure, which takes approximately 2 min, while displaying status windows (Figure 92). 9. Put the Loading Stage into the wash position. Fig. 92.
Shutdown Once the system is clean, the shutdown procedure will shutdown hardware, fluidics, air will be purged, rinsing and backflushing of the sample line, rinsing and backflushing the sample probe and lastly clearing the waste catcher (Figure 93). The user will then be logged out and if automatic startup has been entered the countdown will begin. Fig. 93. Shutdown Status windows. . IMPORTANT! It is advised to clear waste and refill fluidic containers at the end of the day’s use.
header 9 Automatic Startup The ProSort™ can schedule an automatic startup of the system. Upon automatic start up, the system will verify the stream is coarsely aligned and no clogs are present. If these conditions are not met, the stream will be shut off automatically 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 startup. 1.
Automatic Startup Fig. 95. Automatic Restart window during the shutdown procedure. Fig. 96. Automatic restart window during the decontamination procedure. 9.2 Previously Scheduled Automatic Startups After double clicking the ProSort Software icon on the desktop, a login window will appear. If a previous user has scheduled an automatic startup of the system, a countdown will appear at the bottom of the login window (Figure 97).
9.2 Previously Scheduled Automatic Startups When a user logs onto the system and an automatic startup time is designated, the software will display the Auto Startup Scheduled window (Figure 98). There are three options that can be performed at this time. These options determine whether the automatic startup will proceed as scheduled or be canceled. Fig. 98. Auto Startup Scheduled window. WARNING! Canceling a previously scheduled automatic startup may influence other users’ plans.
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10 Maintenance Always follow the PPE guidelines relevant to your laboratory’s safety procedures for dealing with the chemicals recommended below. 10.1 General Maintenance 10.1.1 Daily It is recommended that the sample line is cleaned every day prior to shutdown. See Section 10.6, Disinfectants, for details regarding approved cleaners for this tubing. The system must be shut down through the software on a daily basis.
Maintenance 10.2 Dealing with Clogs A clogged or partially clogged nozzle tip can occur while analyzing or sorting and it is usually a result of an unfiltered sample delivering clumps of cells to the tip. When this happens, the stream will be unable to spray straight or will be blocked completely. Several things can be done to remove this clog. These options, in order of severity are listed below. Once complete, click the startup button and allow the system to verify that the clog has been removed. 10.2.
10.4 Swap Nozzle Tip Wizard 10.4 Swap Nozzle Tip Wizard The Swap Tip wizard will walk through each step necessary to perform a successful nozzle tip swap. Read through the wizard windows carefully and follow instructions on each screen as you proceed with the protocol. Clear an area around the instrument to have adequate working space. At times, adjustment to the nozzle stage is necessary. Handle the nozzle with clean gloves. First, determine which nozzle system is installed in your cell sorter (Table 29).
Maintenance Fig. 100. Nozzle swap wizard window. 7. Place a lint-free 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-ring that will be removed as well. 9. Pull out the small white nozzle tip. If sonication is desired or necessary: 10. Place the tip into a tube containing DI water. 11. Cap the tube. 12. Place the tube into a sonicator for several minutes. 13.
10.4 Swap Nozzle Tip Wizard Fig. 101. Nozzle swap wizard window. Fig. 102. Nozzle swap wizard window. Fig. 103. Nozzle swap wizard window.
Maintenance 20. Check to see if the stream is hitting the waste hole in the back of 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 bottom right gimbal moves the nozzle foward and back. 22. Fine movements can be made using the gimbals and a hex wrench provided in the accessory kit. Fig. 104. Nozzle swap wizard window.
10.4 Swap Nozzle Tip Wizard Fig. 106. Nozzle swap wizard window. An image of the stream alignment is shown with the stream camera. It is necessary to center the stream and optimize the amount of light 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 the brightest. Once optimized, the wizard arrows will disappear.
Maintenance Figure 108 displays the stream image (left, center) and the nozzle camera image (right). The bottom of the nozzle will be displayed as a double dashed green line. 26. Lower or raise the nozzle, using the three gimballs, until the orange line and the yellow line meet and turn green. The S3 Cell Sorter will again use the micromotors to align the stream to the pinhole and the lasers. The three camera views will be shown again. The micromotors will bring the stream into focus again.
10.4 Swap Nozzle Tip Wizard Fig. 110. Adjust Stream Position window pinhole view Fig. 111. Calibrating Droplets. Fig. 112. Calibrating Droplets.
Maintenance Fig. 113. Quality Control Process. 10.4.2 Nozzle Tip Wizard with the AutoGimbal System The AutoGimbal system uses the software to enable hands-free alignment and optimization of the stream to optics. This provides increased repeatability and eliminates the need to make delicate adjustments by hand. The AutoGimbal system uses five axis picomotors to align the stream to the pinhole and to the lasers. Small micrometer movements are controlled by the software.
10.4 Swap Nozzle Tip Wizard A B Fig. 114. AutoGimbal Swap Tip window. Fig. 115. AutoGimbal Swap Tip window.
Maintenance Cleaning the nozzle tip: The nozzle tip may be clogged visibly, but there may also be a clog that is not visible. A syringe wash may solve the problem. Often the tip will need to be sonicated to remove micro clogs or stuck debris. In some instances the cleaning may need to be repeated several times to resolve the issue. Sonication: 8. Place the tip into a tube of DI water. 9. Cap the tube. 10. Place the tube into a sonicator for 10–30 min. 11. Perform a syringe wash after sonication.
10.4 Swap Nozzle Tip Wizard 20. Remove the lint-free cloth from nozzle chamber. 21. Press the nozzle stage back down. The nozzle stage should snap back into position (Figure 117). 22. Click the blue forward arrow. Fig. 117. AutoGimbal Swap Tip window. 23. Slide the sort chamber door open. 24. Clean the sort chamber to ensure there are no stray droplets on the back wall, sort tray, or deflection plates, and that the streams camera window is clean (Figure 118). 25.
Maintenance Fig. 118. AutoGimbal Swap Tip window. Fig. 119. AutoGimbal Swap Tip window.
10.4 Swap Nozzle Tip Wizard Fig. 120. AutoGimbal Swap Tip window. 26. If after reseating the nozzle tip the stream is still hitting the back wall, sort tray, or deflection plates, proceed to step 28. 27. Click the Stream Adjustment icon (see bottom of Figure 119). 28. The Adjust Stream Position window will open (Figure 121). Fig. 121. Adjust Stream Position window.
Maintenance 29. Using the Pitch and Roll arrows, adjust the stream’s position until the stream is hitting the waste hole. 30. Click the blue forward arrow. 31. Close the sort chamber door. 32. Using the Pitch and Roll arrows, move the stream until it is visible as a vertical white line in the camera image on the right of the window (Figure 12). 33. Adjust the stream so that the red dotted lines become green. 34. Click the blue forward arrow.
10.4 Swap Nozzle Tip Wizard Fig. 123. Adjust Stream Position window. 38. The AutoGimbal system will commence automatic alignment of the nozzle tip and stream to the optics. During the automatic alignment process, a series of windows will appear (Figure 124). This process can take 5–6 min to complete. Fig. 124. Automated alignment process windows.
Maintenance Fig. 125. Automated alignment process windows. After the AutoGimbal system has completed its alignment and found the most optimal position, system calibration and drop delay calculation will begin automatically. 39. Select the bead lot from the dropdown menu and click the checkmark (Figure 126). 40. Load 500 µl ProLine™ Universal Calibration or ProLine Calibration Beads into a 5 ml tube and place into the sample station. 41. Move the loading stage into running position. Fig. 126.
10.5 Optical Filter Cleaning 10.5 Optical Filter Cleaning The optical filters in the S3 and S3e Systems will lose performance when dirt, dust, or fingerprints are present on the glass surface. Inspection of these optical components and regular cleaning will help maintain the high performance of the system. Always remember that these coated pieces of glass are delicate and should be handled with care. Any scrape or scratch on the surface could significantly affect the light passing though.
Maintenance It is recommended to run the decontaminate procedure at least once every six months but can be run as often as monthly. This tool should also be used if there is a noticeably high background level in the acquired data. The source could be within the fluidic path. Bacteria or fungus can potentially grow in the lines if samples are not handled using basic cellular sterile technique. The bulk fluidics can also contribute to contamination, despite having internal filters built into the lines.
10.7 Decontamination 8. Replace the DI water and sheath fluid containers with at least 1 L of decontamination solution (Figure 130). 9. Empty waste container and replace. 10. Click the checkmark button to proceed. Fig. 130. Decontamination instructions to prepare the system. The system will drain the internal sheath reservoirs into the waste container (Figure 131). The system will directly prime the fluidics with the decontamination fluid (Figure 132). Fig. 131.
Maintenance In order to decontaminate, the system will soak the fluidic lines in the decontamination fluid and then will shut down (Figure 134). Fig. 134. Decontamination window during the soaking period and shutdown. After the system shuts down, the containers can be removed and the sheath and DI water cap assemblies should be rinsed with DI water. Sheath fluid and DI water containers can be returned to proper position (Figure 135).
10.7 Decontamination Fig. 137. Decontamination window when priming rinse filter and nozzle path. Note: The user must be present to change the containers approximately 1 hour into the procedure. After this point the system can be left overnight to complete the rinse and shutdown if that option is chosen. The system will soak and go through an extended warmup (Figure 138) before a system startup is commenced (Figure 139). Fig. 138.
Maintenance After decontamination the system should be ready for running samples. Any high background should be eliminated. Fig. 139. Startup process at end of decontamination.
11 Troubleshooting Table 30 describes possible errors and causes with troubleshooting steps. Table 30. Troubleshooting table.
Troubleshooting Table 30. Troubleshooting table (continued).
Troubleshooting Table 30. Troubleshooting table (continued). Error Possible Causes Troubleshooting Steps High event rate (while trying Drop drive noise causing Toggle between the FSC/SSC trigger (in the to run QC, or running a excessive event rate service panel) sample at 500 eps) (>40,000 eps) Perform a nozzle tip swap Calibrate droplets (located in the Edit Droplets window when logged in as administrator) Light leak in system, ensure all covers are closed Too much light.
Troubleshooting Table 30. Troubleshooting table (continued). Error Possible Causes Troubleshooting Steps Decontamination procedure Software error freezes during priming Contact technical support for help on the phone. Stream is spraying everywhere Run nozzle tip swap wizard and install nozzle. No tip in place No O-ring in place Run nozzle tip swap wizard to reseat nozzle with O-ring. Tip is still clogged Run nozzle tip swap wizard to clean nozzle using sonication and syringe wash.
12 References The following references and resources are useful for learning more about flow cytometry and its applications. Shapiro HM (2003). Practical Flow Cytometry: Fourth Edition (Hoboken: John Wiley & Sons). Cytometry: Part A; Journal of the International Society for the Advancement of Cytometry. Wiley. http://onlinelibrary.wiley.com/journal/10.
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13 S3 and S3e™ Cell Sorter Specifications ™ The specifications for the S3 and S3e Cell Sorters are shown in Table 31. Table 31. S3 and S3e Cell Sorter specifications.
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Ordering Information Catalog # Description Instrumentation 145-1001 S3 Cell Sorter (488 nm) 145-1002 S3 Cell Sorter (488/561 nm) 145-1005 S3e Cell Sorter (488 nm) 145-1006 S3e Cell Sorter (488/561 nm) 145-1078 S3 Biosafety System Class I 145-1021 S3 Cell Sorter (488 nm), with S3 Biosafety System Class I 145-1022 S3 Cell Sorter (488/561 nm), with S3 Biosafety System Class I 145-1029 S3e Cell Sorter (488 nm), with S3 Biosafety System Class I 145-1030 S3e Cell Sorter (488/561 nm), with S3 Bi
Bio-Rad Laboratories, Inc. Web site www.bio-rad.
