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BD Accuri C6 Flow Cytometer Instrument ManualInstall the software on the computer before connecting the USB cable tothe cytometer.NOTE: We recommend making the following changes to the C6 workstationcomputer in order to achieve the best possible work environment and to avoidunexpected software computer issues that could potentially result in a loss of data.3.2.2Changing the Workstation Computer Settings for Windows 7Skip this section if your computer has BD Accuri C6 Software preinstalled.1. Go to Start (circular icon located in the lower left-hand corner) / select ControlPanel.2. Select “Appearance and Personalization”3. Turn off the Sleep Settings.a. From the column on the left, select “System and Security” then “PowerOptions”b. Select “Change when the computer sleeps” from the left hand margin(alternatively select “change plan settings” from the main window).c. From the Edit Plan Settings window, change the “Put the computer to sleep”setting to “Never” using the drop-down function (we also suggest setting the“Turn off the display” setting to Never).d. Select “Save Changes”. Then close the control panel window.4. Optimize Monitor Display Settings.(From Start / Control Panel / Appearance and Personalization)a. Select “Display”, select “Adjust Resolution” from the column on the left.(Alternatively select “Adjust screen resolution” directly from the Displaysection of the main Appearance and Personalization window). Then select(highlight) Monitor 1.b. Ensure the Resolution is set to the maximum setting or to at least 1152 x 864.Change the setting using the slider under the “Resolution” field’s drop downmenu, if necessary.c. Select “Apply”, then select “OK” and close the Personalization window.To view hidden file extensions which distinguish BD Accuri C6 Software files (.c6)from BD Accuri C6 Software template files (.c6t):1. Click on the Windows Start button and select Control Panel from the pop-upmenu.2. Select “Tools” from the menu bar.3. Click on “View” tab4. Click “Show hidden files and folders”5. Deselect “Hide extensions for known file types”67820018-01 Rev-2

BD Accuri C6 Flow Cytometer Instrument Manual6. Select “Apply,” then “OK” to save changes and close dialog box.1. Remove the cytometer and other components from the shipping boxes.2. With the front of the cytometer facing toward you, lift the top cover.3. Verify that the in-line sheath filter is installed. If it is not installed, see section 4.11for instructions on replacing the filter.Figure 3-2. In-line Sheath Filter4. Verify that there are no disconnected electronic cables on the optics assembly.5. Verify that the interference filters are firmly seated (Figure 3.3). The standardinterference filters are labeled as follows: FL1—533/30 FL2—585/40 FL3—670LP FL4—675/25Interference filterFigure 3-3. Interference FilterNever run the cytometer without interference filters installed. Running thecytometer without the filters may damage the instrument.7820018 Rev-27

BD Accuri C6 Flow Cytometer Instrument Manual6. Gently close the cytometer cover. Make sure the cover is securely positioned. Ifthe cover is not closed securely, the cytometer will not function.7. Place the fluidics bottle tray in a convenient location near the perimeter of thecytometer.Do not place the fluidics bottle tray on top of the cytometer.1. Fill the sheath bottle with sheath fluid.2. Dilute the decontamination concentrate (PN 653154) and cleaning concentrate(PN 653157) solutions as instructed and fill decontamination and cleaner bottles,respectively.Close the bottles securely. Do not over tighten.3. Add approximately 100 mL of 0.5% NaOCl to the Waste bottle.4. Place the bottles in the fluidics bottle tray.Fluidics harnessFigure 3-4. Fluidics harness5. Slide the red tab on the black end of the fluidics harness to the unlock positionand attach the black end of the fluidics harness to the back of the cytometer.6. Slide the red tab to the lock position.7. Attach the other end of the fluidics harness to the appropriate bottles: Red line—waste bottle Blue line—sheath bottle Yellow line—decontamination bottle Green line—cleaner bottle87820018-01 Rev-2

BD Accuri C6 Flow Cytometer Instrument ManualFigure 3-5. Proper Attachment of Fluidics Lines1. Plug the cord from the power supply into the back of the cytometer.Power jackUSB portBD CSampler serialcable (used with BDCSampler only)Figure 3-6. Cable Connections2. Attach the black power cord to the power supply and plug into a standard outlet.Only operate this instrument from a supply source that incorporates athird-wire protective grounding conductor. A surge suppressor or power strip isrecommended.3. Connect the USB cable to the back of the cytometer.Do not plug the USB cable into the computer yet.To prevent putting pressure on the fluidics lines and USB cableconnection, do not place the cytometer against a wall or another object.7820018 Rev-29

BD Accuri C6 Flow Cytometer Instrument ManualThis section provides details on installing BD Accuri C6 Software and the BD AccuriC6 flow cytometer driver. In most cases, BD Accuri C6 Software will be pre-installed.If software is installed, skip to section 3.5.1. Ensure the USB cable is not connected between the computer and cytometer.2. Start up the computer and monitor.3. Insert the provided BD Accuri C6 Software CD. In the CD directory, double-clickon BD Accuri C6 Software Installer.exe.4. Follow the instructions in the installation wizard.1. Check fluid levels in all bottles, ensuring that the Waste is empty and the Sheath,Cleaner and Decontamination bottles are full.2. Gently push the sample stage back, remove and discard the SIP cover and placea tube containing at least 2 mL of 0.22 µm filtered, DI water on the SIP.SIP CollarSIPSample StageFigure 3-7 SIP and Sample StageNOTE: The sample stage can accommodate several types of tubes, includingany brand of 12x75 mm tube and most microcentrifuge tubes3. While holding the sample tube in place, pull the sample stage forward to supportthe tube. The stage clicks into place.The sample tube does not form a seal at the top of the SIP. Whileinserting the SIP in the tube, be careful not to bend or catch the SIP on theoutside of the tube.107820018-01 Rev-2

BD Accuri C6 Flow Cytometer Instrument Manual4. Firmly press the power button on the front of the unit to turn on the cytometer.During start up, the BD Accuri C6 Software Traffic Light turns yellow and thecytometer pumps start to run. When powered on, the cytometer automaticallyflushes the fluidics lines with sheath. This process takes approximately fiveminutes.Do not open the lid of the cytometer during the startup process. Openingthe lid interrupts the laser warm-up and extends the time before data can beacquired.5. When the BD Accuri C6 Software Traffic Light turns green and BD Accuri C6Software displays the message C6 is connected and ready, run 0.22 µm filtered,DI water for at least 15 minutes before processing samples.6. Validate cytometer performance. See the BD Accuri C6 Software User’s Guidefor details on instrument validation using the validation beads provided with thecytometer.1. Place a tube with 2 mL of decontamination solution on the SIP.2. Select an empty data well in the Collect Tab of BD Accuri C6 Software.3. Set the time limit for two minutes and the fluidics speed to fast.4. Click on the RUN button.5. Once the run is finished, remove the tube of decontamination solution from theSIP.6. Place a tube with 2 mL of 0.22 µm filtered, DI water on the SIP and advance toany empty data well.7. Set the time limit for two minutes and fluidics to Fast.8. Click on the RUN button.9. When the run is finished, leave the tube on the SIP.10. Press the power button. The shutdown cycle runs for approximately 15 minutes,then the cytometer automatically powers off.Depressing the power button for five seconds or longer bypasses the autofluidic shutdown cycle. If the cytometer is shut down in this manner, BD AccuriC6 Software displays the following message during the next start up:Figure 3-8. Improper Shut down MessageWhen this occurs, the cytometer takes additional time to recover and return tothe green light ready state.7820018 Rev-211

BD Accuri C6 Flow Cytometer Instrument ManualNOTE: It is not necessary to exit BD Accuri C6 Software or turn off the computerbefore turning off the cytometer.127820018-01 Rev-2

BD Accuri C6 Flow Cytometer Instrument ManualChanging the in-line sheath filter, fluidic bottle filters, and peristaltic pump tubingevery two months is recommended. Heavy use of the cytometer may warrant morefrequent changes.The following table lists the parts and reagents that are required for regularmaintenance.Table 4-1. BD Accuri C6 Flow Cytometer Replacement Parts/Reagents ListPart 3156653157653158653159653160DescriptionPeristaltic Pump Tubing (Set of 2)Sheath bottle filter (1 large) and Decon/CleanerBottle Filters (2 small)In-line Sheath Filters (1 ea.)BD Accuri C6 Flow Cytometer 1-yearMaintenance Kit contains: (12) Peristaltic PumpTubing, (6) Sheath Bottle Filter, (12)Decontamination/Cleaner Bottle Filters, and (6)In-line Sheath FiltersBD Accuri C6 Flow Cytometer 2-MonthMaintenance Kit contains (2) Peristaltic PumpTubing, (1) Sheath Bottle Filter, (2)Decon/Cleaner Bottle Filters and (1) In-lineSheath FilterDecontamination Concentrate Solution (makes200 mL)Decontamination Concentrate Solution (makes1 L)Bacteriostatic Concentrate Solution (for treating10 L Sheath Fluid) (10 vials)Cleaning Concentrate Solution (makes 1 L)BD Accuri C6 Flow Cytometer Fluid Kit:contains concentrate for making 1 L each ofCleaning and Decontamination Solutions,Bacteriostatic Concentrate for treating 10 L ofSheath, and 8 mL of Extended Flow Cell CleanExtended Flow Cell Clean SolutionBD Accuri C6 Flow Cytometer Starter Kit:Contains concentrate to make 1 L each ofCleaning and Decontamination Solutions,Bacteriostatic Concentrate to treat 10 L ofSheath,8 mL of Extended Flow Cell Clean, and4 mL of both 6- and 8-Peak Validation BeadsVisit www.accuricytometers.com for a complete list of available parts and reagents.An automated notification and reminder system is contained in the BD Accuri C6Software to alert the user to perform the recommended preventive maintenance asdescribed in this manual. This includes removal and replacement of the peristalticpump tubing, in-line sheath filter and the fluidic bottle filters.7820018 Rev-213

BD Accuri C6 Flow Cytometer Instrument ManualBD Accuri C6 Software will trigger the notification based on the volume of sheath runthrough the system or on the date since maintenance was last performed. BD AccuriC6 Software will display this notification based on whichever trigger is reached first.The notification appears as a pop-up window that allows the user to choose between‘Remind me later’ or ‘Maintenance is complete’.Selecting ‘Maintenance is complete’ will re-start both triggers.Selecting ‘Remind me later’ will set the notification to remind the user in 5 days.Figure 4-1. Automated Preventive Maintenance Notification WindowRun the backflush cycle as needed to clean the SIP and remove clogs at the base ofthe SIP.1. Place a blotter or empty sample tube under the SIP to catch dripping fluid.2. Do one of the following: Click on the Backflush button in the Collect tab. Select Instrument Run Backflush CycleClean the flow cell as a part of regular maintenance or to correct performance issuesof the cytometer.The unclog cycle purges the flow cell of debris.1. Remove the sample tube from the SIP and place a blotter or empty sample tubeunder the SIP to catch dripping fluid.2. Do one of the following:147820018-01 Rev-2

BD Accuri C6 Flow Cytometer Instrument Manual Click on the Unclog button in the Collect tab.Select Instrument Run Unclog Cycle.For a thorough cleaning of the flow cell, perform an extended flow cell clean. Duringextended flow cell cleaning, the flow cell fills completely with cleaning solution fromthe sample tube on the SIP. This cycle automatically shuts down the cytometer withcleaning reagent in the flow cell, allowing the flow cell to soak.1. Place a tube with at least 500 µL of Extended Flow Cell Clean Solution (PN653159) on the SIP.Never run the Extended Clean of Flow Cell cycle without a tube containingat least 500 µL of fluid.2. Select Instrument Extended clean of flow cell.3. After the cytometer is automatically shut down, allow the cytometer to rest for atleast 30 minutes (up to overnight for a more thorough cleaning).4. Replace the tube of Extended Flow Cell Clean Solution with a tube of 2 mL 0.22µm filtered, DI water.5. Restart the cytometer. The cytometer performs a longer fluidics startup cycle andthe Software displays the message Extra startup time needed due to cleaning orimproper shutdown. This longer cycle purges cleaning reagent from the flow cell.6. Run 0.22 µm filtered, DI water for 10 minutes on fast speed.7. Operate the cytometer as usual.The BD Accuri C6 automatically runs the decontamination fluid cycle when thesystem is shut down normally. The decontamination fluid cycle lasts about 15minutes.To manually run the decontamination fluid cycle:1. Place a tube with 2mL of 0.22 µm filtered, DI water on the SIP.2. Select Instrument Run decontamination fluid cycle.The cleaning fluid cycle pulls cleaner fluid from the cleaner tank and runs it throughthe fluidic lines of the fluidics system. After filling the system with cleaner fluid, thecleaning fluid cycle purges the cytometer with fresh sheath fluid and performs abackflush. This cycle takes about five minutes.1. Place a tube of cleaning solution (PN 653157) on the SIP.2. Select Instrument Run cleaning fluid cycle.7820018 Rev-215

BD Accuri C6 Flow Cytometer Instrument ManualPurge air from the cytometer if any of the following is true: The cytometer is being set up for the first time. The cytometer has not been used for more than two days. A sample has been run dry or the cytometer has been running without sheath inthe sheath bottle.To purge air from the cytometer:1. Place a tube containing sheath on the SIP.NOTE: If performing an air purge at any time other than initial setup, skip steps2-3.2. Select Instrument Run decontamination fluid cycle.3. Wait approximately 15 minutes for the decontamination fluid cycle to complete.4. Set the run time in the software control panel to five minutes and click on the Runbutton. Allow the cytometer to run until it stops automatically.NOTE: The peristaltic pumps will make a rhythmic sound while running. This isnormal.5. Click on the Run button again, wait for 30 seconds, and click on the Pause buttonto stop the pumps.6. Repeat step 5 two more times to purge bubbles from the system.Empty the waste bottle daily or when prompted by BD Accuri C6 Software to preventspillover.Biological samples are potentially dangerous and/or life threatening. Adhere toproper handling procedures for samples and reagents at all times. Wear appropriatelaboratory attire such as protective gloves and eyewear during this procedure.1. Disconnect the quick connect waste line from the top of the waste bottle.167820018-01 Rev-2

BD Accuri C6 Flow Cytometer Instrument ManualWaste line quick disconnectFigure 4-2. Waste Line Quick Disconnect2. Remove the bottle lid.3. Dispose of waste according to local regulations.4. Add approximately 100 mL of 0.5% NaOCl to the Waste bottle.5. Replace the lid.6. Reattach the waste line to the top of the bottle.Before turning on the cytometer, visually check all the bottles and fill the sheath,decontamination, and cleaning solution bottles as needed.NOTE: The cytometer is a non-pressurized system. If necessary, any of the bottlescan be opened while the cytometer is powered on.NOTE: BD Accuri C6 Sof

The BD Accuri C6 flow cytometer offers an entirely new vision for the role of flow cytometry in life science research. Designed from the ground up to be compact and easy to use, the BD Accuri C6 flow cytometer fits in any lab and is readily accessible to all researchers—experts and novices alike. Figure 1-1. The BD Accuri C6 Flow Cytometer .