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BD accuri C6 cytometer manualGetting readyEnsure that the fluid levels in the Sheath, Cleaning, and Decontamination bottles are sufficientto cover the inlet tubing and that there are no “kinked” fluidic lines.Turn on the computer and log in with your UCF info. Start the Accuri CSampler software.Press the Power button on the BD Accuri C6 instrument face panel. The instrument will begin a5 minute auto start cycle. If the BD Accuri software displays the message Extra Start-up timeneeded due to cleaning or improper shut down it will take the instrument several more minutesfor the instrument to get to the green-light ready state. Dripping from the SIP at the beginningof the Auto-Start cycle is normal.When the instrument is ready, the traffic light within the workspace will turn from yellow togreen. It is recommended that you install a tube of DI water and run in Fast mode or custom100µl/minute mode for several minutes to verify fluid uptake and check for contamination withparticles. If you are the first user of the day, you can run for 15 minutes; by then the lasers willbe warmed up. Handle the SIP and Sample Support Arm Gently! It is also recommended topress the Backflush button and collect flow-out on kimwipes or into a tube (ca.100-200µl). Theaccuri is now ready.If doing rare event detection or working with sticky samples, it is recommended that aBackflush is run between samples. Wiping the SIP and back flushing between samples willprevent carryover between samples.Setting Fluidics RateThe system can accommodate an upper limit of 10,000 events per second, but it isrecommended to acquire samples at a rate of 2,500 events per second or less to ensure thebest data resolution and prevent problems with clogging.To set the fluidics rate: Select from the Slow(14µl/min), Medium(35µl/min), or Fast(66 µl/min)button in the Fluidics section of the Collect tab.Threshold

Use thresholds to eliminate light scatter and/or fluorescence signals caused by debris insamples and electronic noise inherent in the system. By default, BD Accuri C6 Software is set toa primary threshold of channel 80,000 on FSC-H. Use the THRESHOLD button on the workspace.Threshold settings can be changed before, during, or after data acquisition. All thresholds areset on the Height signal for any given parameter. Width parameter is based on Threshold parameter settingFSC Threshold should be set from 200K-500K for most mammalian cells with theexception of very large cells 0.5M-1.0M or platelets (10K-30K). Begin at a value low inthe range and raise as needed.Setting a Run LimitThe run limit defines when data acquisition will stop. Time, Volume, or Number ofEvents in a specified gate are used to define the run limit. If multiple run limits are set,data collection stops on whichever limit is reached first. If the Run Unlimited check boxis checked the sample will not stop running until 1 million events (the maximum allowedper sample) are collected.Creating and Editing PlotsCreate a plot(s) using the buttons located in each designated plot area in the Accuriworkspace. Plots cannot be resized.Use the Plot Spec Tool found in each plot to manipulate the data display in a plot,including axis parameter selection, channel range specification, and selection of linearor logarithmic axis scale. Set up or modify plot specifications at any time before or aftercollecting data.To modify the event number to be displayed, Select Display Events Display Settingsfrom the top menu. Choose from All, custom number or custom percentage.To re-name plot axis labels, click on a plot axis and Choose Rename Parameters fromthe dropdown menu. Enter a new name in the Rename to field.StatisticsWhen a plot is selected, statistics for that plot and any gates drawn in that plot willshow up at the top of the plot analysis area. You can select for Median Statistics to berevealed or hidden at anytime by clicking the Display tab Show Median Statistics.Gating

There are a number of gating types available. These include quadrant, rectilinear, andpolygonal for 2-parameter plots and vertical and horizontal markers for histograms. BDAccuri C6 Software automatically displays the percentage of cells within the region. Gates cannot only be deleted from the workspace by deleting the plot on which it wasdrawn. Gate names are assigned sequentially by type (R1, R2 R3, P1, P2, P3, M1, M2etc). as created.Custom gate names can be entered by double-clicking on the label and typing a newgate name in the dialog box.CompensationCompensation is the process used to correct for the spillover of fluorescence into the wrongdetector(s). On the Accuri C6, all compensation is performed manually. Post- acquisitioncompensation can be performed using Accuri C6 software or third-party applications.To perform compensation,1. Open the Compensation Template from the desktop2. Collect at least 500 positive events for each single-stain control3. Adjust compensation manually using the Set Color Compensation dialog box. In the rowassociated with the detector to correct, click on the FL button of the fluorochromechannel that is creating the spillover. Adjust the value while monitoring the quadrantstatistical means for the plot containing both FL readouts. Repeat this with allfluorochrome detector combinations in the experiment.4. Apply the compensation values to all sample tubes.Clogs/ ObstructionsThe BD Accuri C6 has a 1-minute UNCLOG cycle which may be run any time an obstruction issuspected. Place an empty tube on the sample port and select the UNCLOG button in theworkspace. Fluid should accumulate in the tube. The cycle may be repeated until fluid isobserved in the tube.Exporting DataData can be exported from sample wells as individual FCS data files. Files will automatically besaved to a folder named EXPORT on the desktop. Transfer a copy of the files to your UFCRserver space and move them to your lab folder in the My Documents folder. Choose File Export FCS File. Delete any old data from your folder at each visit.

Basic features of the BD accuriCleaning the instrument after use1. When Cytometer is at green‐light ready state, place a paper towel ortube underneath the SIP. Click BACKFLUSH.2. Clean the “home position” of the SIP, i.e. the white receptacle on theplate platform: put Kimwipes around the “home position” and spraysome water into the opening. Dry excessive water with more Kimwipes.3. Place 4 tubes into the 24 tube rack: a tube with 2 mL of dilutedDecontamination Solution, a tube with 2ml diH2O, a tube with 2 mL ofdiluted Cleaning Solution and another tube with 2 mL diH2O on the 24tube rack. Select the data wells. Set time limit for 2 minutes and Fluidicsspeed to Fast. Click RUN The Cytometer will stop automatically when thetime limit is reached. Leave the tubes on the 24 tube rack until theCytometer is used again.4. Switch instrument off if nobody else will use it the same day. The BDAccuri C6 automatically runs a fluidics system shutdown. Depress thePower button on the Accuri instrument face panel for 1 second and theinstrument will auto shutdown. Once the shutdown cycle has begun(Flashing blue light on front instrument panel) the computer can bepowered off.5. Please logoff Windows before you leave.

Taking care of fluids:Empty the waste tank daily or when prompted by CFlow software to prevent spillover andpossible biological safety risk.1. Disconnect the waste level sensor and quick disconnect from the top of the Waste Bottle.2. Remove the lid.3. Dispose of waste according to local regulations.4. Add approximately 100 mL of 0.5% NaOCl to the bottle.5. Replace the lid.6. Reattach the waste level sensor and quick disconnect to the top of the bottle.Filling the Sheath, Decontamination, and Cleaning Solution BottlesBefore turning on the C6 Flow Cytometer, visually check all the bottles and fill the Sheath,Decontamination, and Cleaning Solution Bottles accordingly (instructions on how to preparethe solutions are found on the stock solutions).1. Disconnect the appropriate level sensor and quick disconnect from the top of the bottle.2. Remove the bottle lid.3. Fill the bottle with appropriate reagent.4. Replace the lid.5. Reattach the level sensor and quick disconnect to the top of the bottle.The C6 Flow Cytometer is a non-pressurized system. If needed, any of the bottles can beopened while the C6 is powered on.AreaSampleconcentrationCell suspensionSample mediumSample typeRecommendations1,000–5 x 106 cells/mLAssess and minimize cell clumping.Calibrate fuidics when necessary to account for liquid viscosity.Cell lines, Primary cells, Beads, Bacteria12 x 75-mm tube: 300 μL–2 mLSample volumeUsers should verify other tube/plate types, calibrate fuidics when necessary.Standard settings: Medium or FastCustom settings: Minimum settings are listed below. Appropriate flow rateFluidics speedand core size combinations are experiment specific and should be validatedby the user.Sheath fluid0.2µM filtered water with bacteriostatic added.

Recommended preventive maintenance procedures:FrequencyAfter everyexperimentDaily (if thecytometer is notshut down)Between usesMonthlyBi-monthlyTaskRun filtered DI water at the Fast rate for 3 to 5minutes to prevent clogging.Run the decontamination and cleaning cycles.Place a tube of DI water on the Sample IntroductionProbe (SIP), or if using the BD CSampler option,place the SIP in the wash station.Clean the flow cell by performing an extended flowcell clean.Replace the peristaltic pump tubing, in-line sheathfilter, and bottle filters.Supplies and partsDI waterDI water, Cat. Nos.653154, 653155,and 653157DI waterCat. No. 653159Cat. Nos. 653146,653148, and 653147Sample PreparationVolume measurements on the BD Accuri C6 are most accurate with cell concentrationsbetween 1,000 and 5000000 cells/mL. Higher concentrations might result in inaccuratecounting due to system saturation, so dilute samples if necessary. The ideal cell concentrationrange for accurate counting varies among cell types, based on considerations such as size,shape, and tendency to clump. The flow rate, relative to sample concentration, should minimizedoublets and larger clumps, yet never exceed 10,000 events per second. We recommend thatyou test serial dilutions of the sample and compare reported concentrations to verify a linearcorrelation.Assess and minimize cell clumping, either by dilution or a combination of enzymatic andmechanical means. Cells particularly prone to clumping may need to be filtered prior to runningon a flow cytometer. Cells must be evenly dispersed throughout the suspension; otherwise, nomethod can achieve accurate counting.To assess cell clumping plot FSC-A against FSC-H. When single cells in the flow cell pass throughthe laser beam, their FSC-A and FSC-H signals correlate linearly and plot along a relativelystraight line. Clumps of cells will have larger FSC-A signals relative to FSC- H, and the signals willfall off the diagonal formed by single cells. Diluting samples may help to reduce the percentageof clumping cells.

Useful tools for dye/fluorochrome cencespectraviewer.html?gclid CPaFz7T gdICFYEYgQodgywO7w&s kwcid r&ef id WFLtnwAABSMAgxpg:20170209024442:sTable: Settings for various fluorescent dyes

Examples for DNA dyes on the BD accuri:Using the blue/488 laserDNA dyes for the red/640 laser:

Fluorescent proteins on the BD accuri:Blue laser:Red laser:Examples of fluorochromes for the BD accuri:

Blue laserdyes for the 640/red laser:same dyes when activated by the 488/blue laser:

The C6 Flow Cytometer is a non-pressurized system. If needed, any of the bottles can be opened while the C6 is powered on. Area Recommendations Sample concentration 1,000–5 x 106 cells/mL Cell suspension Assess and minimize cell clumping. Sample medium Calibrate fuidics when necessary to account for liquid viscosity.File Size: 787KB