Frequently Asked Questions

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Topics

Capabilities & Performance

Can I perform absolute counts with the BD Accuri C6 flow cytometer?

Yes. The BD Accuri C6 flow cytometer simultaneously measures events (cells) and the volume of sample fluid passing through the system. From this information BD CFlow can calculate the absolute number of events per microliter of sample fluid.

How do I set the voltage gains?

The BD Accuri C6 flow cytometer has a broad dynamic range and does not require the user to manipulate voltage gain settings. All available sample data can be collected with excellent resolution using the factory settings. This feature eliminates a time consuming step in data collection, and greatly reduces the chance that data will be lost.

Can I use PE-Cy5 conjugates on the BD Accuri C6?

Yes. The major fluorescence signal from PE-Cy5 will be detected in FL3 (670 LP) on the BD Accuri C6. Because the Cy-5 portion of this tandem dye is excited by the 640 red laser, there usually is significant fluorescence detected in FL4 (675 BP) as well. The amount of fluorescence compensation needed to subtract PE-Cy5 signal from FL4 is often on the order of 50 to 100%.

Because of this large fluorescence bleedover component, we recommend careful selection of antigen and fluorochrome combinations when pairing PE-Cy5 with detection of other markers in FL4. Highly expressed antigens conjugated to APC or Alexa-647 can work in combination with PE-Cy5, but appropriate single-stained and background fluorescence controls must be run. An alternative approach when simultaneous PE-Cy5 and FL4 detection is required, is to use far red fluorescent probes (e.g. APC-Cy7, APC-H7), switching the standard FL4 filter (675 BP) to a 780 BP (Cat. No. 653187). This will decrease the PE-Cy5 bleedover component into FL4.

Do I need to change settings to collect in log or linear mode?

No. The BD Accuri C6 flow cytometer collects all data in linear mode, and BD CFlow can display the data in linear or log format. The change from linear to log can be made or reversed at anytime during or after data collection. The default display settings for BD CFlow display scatter data in linear format and fluorescent data in log format.

Can I change the optical filters in the BD Accuri C6 flow cytometer?

Yes. The protocol for replacing optical filters is easy and takes a few seconds.

Which signal is measured if I select the Width option from the axis label -down menu?

The Width signal is always for the parameter chosen as the Primary Threshold. The default Primary Threshold parameter when opening a new BD CFlow File is forward light scatter (FSC).

Can I use the FL4 fluorescent detector with the blue laser?

Yes, if the Selectable Laser Module is installed.

What type of sample tubes can I use?

The BD Accuri C6 flow cytometer does not use a pressurized fluidics system, so it is very tolerant of the type and brand of sample tube used. The Sample Tube Shelf can accommodate several types of tubes, including:

12 x 75 mm tubes, any brand
Most microcentrifuge tubes
Other tubes not larger than 16x78 mm

Can I sort cells with a BD Accuri C6 flow cytometer?

No. The BD Accuri C6 flow cytometer is an analytical cytometer. The test sample (cells) is discharged into the waste tank after analysis.

Software & Data

Do I need to load BD CFlow software on the computer?

No, the computer supplied with the unit has BD CFlow installed. You may purchase additional copies of BD CFlow to load on your own computer(s) running Windows XP or greater.

Can I use BD CFlow software on a Mac?

The BD Accuri C6 flow cytometer system cannot be operated using a Mac, however a stand-alone version of BD CFlow Analysis is available for Mac.

Can BD CFlow read data produced on another manufacturer's cytometer?

No. BD CFlow is designed to only work with data collected on the BD Accuri C6. However, BD CFlow can export data as FCS 3.0 files which can be compared to data from other manufacturer's cytometers using 3rd party software programs such as FCS Express and FlowJo.

Can BD CFlow data be imported into other software programs?

Yes. Even though the BD Accuri C6 flow cytometer produces data over 7 decades of dynamic range, most 3rd party analysis programs, such as FCS Express and FlowJo, can open the FCS 3.0 files exported from BD CFlow.

What is the difference between BD CFlow and BD CFlow Plus?

Two software packages are available for the BD Accuri C6 flow cytometer: BD CFlow and BD CFlow Plus. BD CFlow offers all of the cytometer controls and analysis features necessary to run the cytometer, collect data, and analyze results.

BD CFlow Plus contains all the functionality of BD CFlow and has the additional advanced features of user-adjustable, independent fine control of core size and flow rate, the ability to measure and record sample volume, and the addition of the time parameter enabling kinetic measurements of all other parameters.

Operation & Maintenance

How do I clean my BD Accuri C6 flow cytometer?

Generally, all you need to do is turn the instrument off at the end of each day by pressing the power button after placing a tube of DI water on the SIP. The BD Accuri C6 flow cytometer will automatically decontaminate itself. When you turn off the instrument it will run decontamination and sheath fluids through the entire fluidic path. When powered on, the instrument will automatically flush the fluidics lines with sheath fluid prior to reporting that it is ready for a sample. In the case of an unexpected loss of power, the BD Accuri C6 will decontaminate itself during the next power up before reporting that it is ready for a sample.

It is possible to activate this decontamination cycle without turning the BD Accuri C6 flow cytometer off. The Decontamination Cycle is accessible under the Instrument menu. Selecting this option will instruct the BD Accuri C6 flow cytometer to purge the system with the decontamination fluid, followed by a sheath fluid flush.

When shutting down for the day, it is recommended to place a sample tube on the SIP that contains 2-3 mL of sheath fluid.

It is important to ensure that the sheath, decontamination, and cleaning tanks do not run dry and that the waste tank does not overflow. The system is equipped with fluid sensors in each tank to alert the user when they need attention.

Are the cleaning and decontamination fluids toxic?

The Cleaning fluid contains a biodegradable detergent. The Decontamination fluid contains reagent-grade sodium hypochloride. Both can be disposed of down the sink.

What type of sheath fluid should I use?

For most cell analysis we recommend using high grade filtered DI water. Water does not affect the cells during analysis, and it is unlikely to leave salt residue in the fluidics system.

A bacteriostatic additive is available to ensure that the sheath fluid remains clear of contaminants.

How do I know if my BD Accuri C6 flow cytometer is clogged?

Depending on the type and quality of sample used, it is possible for the SIP to clog. The inner diameter of the SIP is 200 microns, so clumps of cells or debris may not be able to enter the fluid path. A clog in the SIP will prevent sample uptake, resulting in a sudden reduction in detectable events. Often a clog can be confirmed by visual inspection of the SIP tip.

How do I clear a clog in my BD Accuri C6 flow cytometer?

To clear a clog in the SIP:

Remove the sample tube.
Click "Backflush" in BD CFlow; the BD Accuri C6 flow cytometer will then run for about 15 seconds. Visually confirm that sheath fluid is dripping from the SIP while Backflush is running. If fluid is not dripping from the SIP while Backflush is running, run Backflush several times in succession until fluid can be seen dripping from the SIP.
If fluid does run out of the SIP during Backflush and yet the BD Accuri C6 still appears clogged, click "Unclog" in BD CFlow; the BD Accuri C6 flow cytometer will then run for approximately 15 seconds. This purges the flow cell.
Check to see if your sample event rate is normal.
The system is now ready to collect.

Do I need to perform scheduled maintenance on the BD Accuri C6 flow cytometer?

Yes. Turning off the instrument at the end of each day, and cleaning the sample injection probe (SIP) regularly are good first steps (make sure the sheath, decontamination and cleaning tanks do not run dry). There are two parts that need to be replaced over time:

Fluid filters (4)
Peristaltic pump tubing (2)

The frequency of replacement depends on usage. We recommend replacement of filters and peristaltic pump tubing every 2 months, or more often as needed. The replacement protocol is easy and can be found in the BD Accuri C6 Flow Cytometer Instrument Manual. Replacement part kits are available.

How do I install the SIP?

To reinstall the SIP Assembly:

Using one hand, push the Sample Tube Shelf back into the Loading Bay, to create space to insert the SIP Assembly.
Using your other hand, carefully place the top of the SIP Assembly into the hole above Sample Tube Shelf. Be careful not to damage the SIP's narrow diameter tip by contacting it with the surrounding plastic or metal.
The SIP Assembly should reach about 1 cm into the hole before it abuts the threads in the hole.
When the top of the SIP is in place, screw the SIP into place. Be careful to not force the SIP into place or strip the threads. It should turn easily.
Stop when it feels tight. The SIP seals with a rubber O-ring; it does not need to be screwed in very tight. Screwing in the SIP too hard can crack the plastic part of the SIP and even damage the cytometer.

Do I need to replace the peristaltic tubing?

Yes, the peristaltic tubing does wear out over time and should be replaced. The frequency of replacement depends on usage. We recommend replacing the peristaltic tubing every 2 months, or more often is needed. The replacement protocol is easy and replacement kits are available.

Does the BD Accuri C6 flow cytometer require special installation?

The BD Accuri C6 flow cytometer is a bench top instrument, and it operates on a universal power supply (100-240 V, 50-60 Hz). There are no special electrical requirements for installation. Additionally, there is no alignment of the optics that needs to be performed after you receive the instrument. Follow the instructions provided in the BD Accuri C6 Flow Cytometer Instruction Manual. Set up should take approximately 60 minutes or less. Then run the provided Calibration Beads to insure that the BD Accuri C6 was not damaged during shipment.

BD CSampler

Can my BD Accuri C6 flow cytometer be upgraded with a BD CSampler?

Yes. If your BD Accuri C6 is already compatible with the BD CSampler, the only additional requirement will be to upgrade to BD CFlow Plus if you currently use BD CFlow software. If your BD Accuri C6 is an older model that is not compatible with BD CSampler then the BD Accuri C6 would need to be replaced with a newer model.

Does upgrading require additional power requirements?

No. The BD CSampler connects to and draws its power directly from the BD Accuri C6. An additional outlet is not required.

How long does it take to install the BD CSampler?

Installation of the BD CSampler takes approximately five minutes. BD CSampler uses three mounting bolts and a single communication/power cable. No additional power outlets are needed as the BD CSampler draws power from the BD Accuri C6. A simple pictorial installation guide is supplied with the system.

Are there any special tools needed for installation?

Yes. A special tool, supplied with the BD CSampler, is used to tighten and/or loosen the mounting bolts. No special experience is needed.

Do I need special software to run the BD CSampler?

The BD CSampler accessory includes BD CFlow Sampler software, used to set up experiments and operate the system. BD CFlow Sampler is based on the easy-to-use BD CFlow Plus so existing BD Accuri C6 owners will be familiar with the look and feel of the interface. Users of BD CFlow will need to upgrade to BD CFlow Plus prior to the installation of BD CSampler and BD CFlow Sampler.

What training is needed for the BD CSampler?

No special training is needed. The BD CSampler kit includes a pictorial Installation Guide and a Quick Start Guide. Detailed information can be found in the BD CFlow User Guide, supplied with every BD Accuri C6, or you can download the CFlow User Guide. BD CFlow Sampler is similar to the BD CFlow Plus platform so it is easy to learn.

Do I need any additional reagents to operate the BD CSampler?

The BD CSampler does not need any additional reagents. Those used with the BD Accuri C6 are compatible with the BD CSampler.

What types of plates can be run on the BD CSampler?

The BD CSampler can accommodate standard 96-well plates (flat, u-, and v- bottom) as well as deep-well 96-well plates. 48-well plates can also be used. 12 x 75 mm tubes can be accommodated, using the 24-tube rack supplied. As some settling of cell/particles may occur over the course of running plates, it is not recommended that v-bottom, flat-bottom and deep-well 96-well plates are used for absolute count determination.

What brands of plates is the BD CSampler compatible with?

There are numerous manufacturers of microplates. All the plates we have tried are manufactured to the guidelines published by the American National Standards Institute, submitted by the Society for Bimolecular Screening.

The following companies were represented in the development of the guidelines: Apogent Discoveries, BD Biosciences, Beckman Coulter, Boehringer-Ingelheim, Co Bio Engineering, Corning, Greiner BioOne, Hamilton Co, Hoffman La Roche, MatriCal, Merck & Co, Millipore, MJ Research, Molecular Devices, REMP, Tecan, Thermo Electron, Whatman

Do I have to remove the BD CSampler to use 12 x 75 mm tubes?

You can continue to process single, or multiple 12 x 75 mm tubes with the provided 24-tube rack. There is no need to remove the BD CSampler.

What about microcentrifuge or bullet tubes?

Currently, these tube types cannot be accommodated by the BD CSampler.

How much extra bench space do I need?

The BD Accuri C6 with BD CSampler takes just a few extra inches of bench space, as compared to the regular BD Accuri C6. It is recommended that the minimum bench depth should be no less than 28 inches (71 cm). The width of the system is 19 ½ inches (49.5 cm).

What is the mat for?

The mat delineates the area of bench space that should be kept clear of obstructions, in order for the BD CSampler to operate safely.

How many different experiments can I run in a single plate?

If desired, different settings could be applied to each well of a 96-well plate! Also, one could use parts of the plate for an experiment and then use the rest of it later. The flexibility of BD CFlow Sampler allows use of single or multiple wells.

How long does it take to process a plate?

With a 30 second acquisition time per well, the processing time for a 96-well plate is less than 90 minutes.

How much sample is left behind in each well (residual or “dead” volume)?

Residual volume varies depending on the plate type however; the residual volume will never exceed 50 µL.

Is there clog detection?

In common with other analytical flow cytometers, there is no specific clog detection on the BD Accuri C6 with BD CSampler. The normal fluidic cycles that occur between samples when performing an automated run will minimize clogs and, in many cases, clear minor clogs. To minimize the impact of a clog on the rest of the plate:

Add a fixed time as one of the stop criteria to prevent the system from aspirating the entire contents of a well.
Add a wash step between samples, with particularly “clumpy” samples, to clear all but the most stubborn blockages.

Do I have to remove BD CFlow Plus when I install BD CFlow Sampler?

No. You do not have to remove BD CFlow Plus from the BD Accuri C6 when the BD CSampler and BD CFlow Sampler are installed, but you cannot use BD CFlow Plus at the same time as BD CFlow Sampler. If the BD CSampler is removed for any reason, BD CFlow Plus is able to read data files generated with BD CFlow Sampler.

Do I need to remove BD CFlow Sampler if I remove the BD CSampler?

No. If the BD CSampler is removed, replace the sample stage and use BD CFlow Plus to control the BD Accuri C6 system.

Will my old BD CFlow/CFlow Plus files be compatible with BD CFlow Sampler?

Yes, since the BD CFlow Sampler was developed on the BD CFlow Plus platform, there are no compatibility issues. Exported files are in FCS 3.0 format.

Do I have to run a Wash after every sample?

If samples are particularly concentrated, one may chose to use the wash function. A single wash will reduce carryover to between 0.1 and 1% and two wash cycles will reduce carryover to less than 0.1%.

Will the Agitate function re-suspend my cells?

The Agitate function is designed to keep cells/particles in suspension, not to re-suspend pelleted samples. A single agitate cycle will maintain fixed lymphocytes from human peripheral blood in suspension (+/-10% of expected value) while processing a 96-well, round-well plate, 30 seconds data acquisition per well. Thoroughly mix samples prior to processing on the BD CSampler.

Can I decide when and where I want the Agitate and Wash cycles to occur during a plate run?

Yes, both Agitate and Wash can be programmed to occur between certain wells or at time intervals during a plate run.

How many events can I collect with the BD CSampler?

With BD CFlow Sampler, each well can contain up to 1 million events. A BD CFlow Sampler file can contain a total of 96 million events.

What about ambient light bleaching my samples?

A plate is exposed to only 15% of ambient light while being analyzed on the BD CSampler. Wrap the plates in general purpose aluminum foil if there is concern over photo-bleaching. The BD Accuri C6 SIP is able to pierce the foil without causing damage to the system.

Is it easy to stop or interrupt a plate after it has been started?

Yes. Plates can be easily interrupted to run other samples, or to modify the plate conditions.

How often do I have to align the BD CSampler?

The BD CSampler automatically aligns itself when it is installed and when it is turned on. If a collision occurs, the software will prompt you to align the BD CSampler. Just click on the command and the alignment process is complete in less than 1 minute.

What is the warranty on the BD CSampler?

1 Year. Extended warranties are available.

What if I have a problem with my BD CSampler?

If you experience a problem with the BD CSampler that cannot be resolved on the telephone by our Technical Support Representatives, we will send you a replacement unit. Once you receive the replacement, return the original BD CSampler to us.