BioFlux One User's Guide

Last updated March 1, 2022

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Table of Contents

Safety and Site Requirements

Safety considerations

If the equipment is used in a manner not specified by Fluxion Biosciences, the protection provided by the equipment may be impaired.

To prevent injury, always observe good laboratory practices when you handle reagents, change tubing, or operate the system.   Know the physical and chemical properties of the reagents you use. Refer to the Material Safety Data Sheets for the reagents in use. Follow a regulated, approved waste disposal program.

To prevent damage to the accessory heater, do not set the temperature controller over 50°C.

Power requirements

The instrument is powered by 12 VDC. Only the universal adapter and power cord supplied with the instrument should be used.

The external adapter permits operation from any line voltage between 100-240 VAC, 50-60 Hz.

For performance meeting the CE Guidelines for Surge Resistance a 220/50 or similar AC inputs, use of an external surge suppressor with the following specifications is recommended:

Surge Suppression Rating: 1020 Joules
Transient Response Time: Instantaneous
Line Noise Suppression Rating: > 75 dB @ 1 MHz

Bench space

The Controller requires bench space 12” (30cm) wide x 13” (33cm) deep and 9” (22cm) tall, not including the computer and monitor. The instrument weight is approximately 30 lbs (13.6 kg). The Controller should be mounted in close proximity to the inverted microscope that will be used for experiments.

Environmental considerations

The user is responsible for operating the BioFlux System in a safe environment.

The instrument is designed to operate in an environment with ambient temperatures between 20-30 °C (68-86 °F) and humidity <95%, non-condensing.

The BioFlux System is not designed to be spark-proof and should not be used in the presence of flammable vapors, gases, or gas mixtures. 

The BioFlux System is designed to bleed off small amounts of the source gas (either ambient air or source tank) as part of the pressure regulation system.

For this reason, toxic gases should not be used with the BioFlux System.

Instrument Installation

To install the system, follow these steps in order:

  • Unpack and inspect the system
  • Assemble the required additional materials
  • Make pneumatic connections

Unpacking and inspecting the system

 To unpack the BioFlux One system:

  • Inspect the shipping cartons for damage or evidence of mishandling. If damage or mishandling is evident, notify the carrier before opening the cartons.
  • Cut the tape and open the flaps on the top of the BioFlux One shipping box. Remove the outer shipping box.
  • Remove the foam packing material from the top of the BioFlux One unit.
  • Remove the cardboard accessory box inside the BioFlux One carton.
  • Carefully lift the BioFlux One System out of the box. Grasp the sides of the instrument.
    A second person may be necessary to help slide the carton and foam inserts off the instrument.
  • Place the instrument on a lab bench in a location with convenient access to power. 

Check the contents of the accessory kit against the parts list (Table 1, below) to confirm that all items are included.

Table 1: Parts List for BioFlux One Shipping Containers

#

Item

Description

Qty

1

Controller

Regulates pressure control and temperature

1

2

Interface

Pneumatic interface; mounts onto BioFlux Plates

1

3

Power Supply

Input: 100-240V 50-60Hz, Output: 12V DC 5A

1

4

Tubing Harness

Connects Controller to Interface

1

5

BioFlux Software

Installation USB drive

1

6

USB Cable

2m length, runs from Controller to USB slot

1

7

Optional Heating Plate

Optional. Connects to Controller, fits BioFlux Plates

1

8

User's Manual

Printed user manual

1

9

Torque Wrench

Used to screw on Interface to plates

1

Visually inspect the system to identify key components

Prior to beginning the installation, check the BioFlux system to identify the location of components, switches, and connections. Refer to the figures below for descriptions.

The Controller is the largest component. It should be unpacked first and set up next to an inverted microscope. The Interface mounts to a BioFlux Plate on the microscope stage (see Fig. 1 for a fully assembled system). The Interface is designed to fit on top of the plates.

Setting up pneumatic connections

About Push-to-Connect Fittings

BioFlux pneumatic connections utilize push-to-connect fittings for easy insertion and removal. To connect the tubing lines, firmly grasp one end of the tubing and push it into the center of the connector. You should be able to feel the tubing engage into the connector, and you can check the connection by pulling on the tube after it is inserted. It should not be able to be pulled out by tugging on it.

To remove the tubing, push the plastic retaining ring in firmly with one hand while pulling on the tubing with your other hand. The tubing should come out easily using moderate pulling force. If it does not come out easily, make sure the plastic ring is being pushed in completely before trying to pull on the tubing. Do not use excessive force to pull out the tubing as this may damage the connectors.

Connect Tubing Harness from the Interface to the Controller

A Tubing Harness is provided which contains 8 tubes bundled together. The 48-well plate interface also has connections for 8 tubes. Note: the BioFlux One controller works with 48-well plates only. 

The picture shown below is the Vapor Trap attached to the BioFlux Controller. The Tubing Harness runs from here to the Interface. Each tube is numbered sequentially corresponding to the port on either end. The pneumatic harness assembly between the BioFlux controller and the Interface is labeled to assist in the correct installation. The Interface end is labeled (1-8 for 48-well plate interface) and plugs into the corresponding numbers on the Interface. The BioFlux controller side of the Harness is labeled using Roman Numerals (I – VIII for 48-well interface).

Connect the tubes to the fluid trap on the BioFlux Controller back panel as follows:

Connect the Tubing Harness to the Interface
Connect the tubing harness to the 48-well plate Interface as follows:

At this point you have made all the required pneumatic connections. 

If you have multiple Interfaces, you can swap between the two by removing the tubing from the fluid trap on the back panel.

Important: the interface gasket is covered with a plastic sheet for protection during shipping. Remove the plastic sheet before use!

Note that the torque driver blades are different for the different interfaces. The 48 well interface uses a 7/64” hex bit.

Setting up electrical connections

There are only three electrical connections to make:

  1. Connect the device end of a USB cable to the back panel socket labeled “USB”, connect the other end to your computer.
  2. Connect the BioFlux power supply output cable to the back panel socket labeled “Power Supply” and plug the other end in to a power outlet.
  3. Connect the optional heater cable connecter to the back panel socket labeled “Heater”

To prevent damage to the accessory heater, do not set the temperature controller over 50°C.

External Gas Supply connections (OPTIONAL)

Note: The Environmental Gas option is intended for use when non-atmospheric gases are desired to be integrated with the system. An internal compressor with a 5-micron particle filter is standard equipment - If no external gas is connected, or if the supply pressure is too low (below 25 psi), the system will operate off of the internal compressor.

To ensure proper operation and to prevent damage to the internal pneumatic connections, set the supply pressure to 25 psi (172 kPa).

  1. Connect the included barbed male fitting to the customer-supplied external gas line. Two fitting sizes are supplied with the unit; 1/8" (3mm), and 1/4" (6mm). It is recommended that a shut-off valve be installed to the gas supply and be turned off when the system is not in use.
  2. Connect the male fitting to the female port on the rear of the BioFlux Controller as shown in the figure.

Software Setup and Functionality

Installing and loading the software module

Installation of the BioFlux software is done by using the provided Software Installation USB drive or software downloaded from the Fluxion knowledge base (https://support.fluxionbio.com/kb/bioflux-200-full-installer).

Ensure that the Windows 10 PC the software will be installed on has internet access during the software installation.

Enable the .NET Framework 3.5 and disable driver signature enforcement

Before installing the software onto a Windows 10 PC, first perform the following:

Enable the .NET Framework 3.5 in Control Panel. You can enable the .NET Framework 3.5

through the Windows Control Panel. This option requires an Internet connection.

Press the Windows key on your keyboard, type "Windows Features", and press Enter. The Turn Windows features on or off dialog box appears. Select the .NET Framework 3.5 (includes .NET 2.0 and 3.0) check box, select OK.

  • Set Windows 10 PC to Ignoring Signature Enforcement at start up by pressing and holding the Shift key on your keyboard and clicking the Restart button.
  • Choose Troubleshoot > Advanced options > Startup Settings and click the Restart button.
  • When your computer restarts, you'll see a list of options. Press F7 on your keyboard to select Disable Driver Signature Enforcement.

Install BioFlux software to run as administrator and Windows 7 compatible

  • Insert the Fluxion USB drive containing the BioFlux Software Installation files. If you no longer have the original USB drive, you can access a downloadable version at https://support.fluxionbio.com/kb/bioflux-200-full-installer.
  • Upon loading BioFlux Full Install V2_5_0_8 (or by manually opening the main directory), a directory of the contents should be displayed on your screen. Right click on “Setup.exe” and choose “Properties”.
  • From the dialog box, click “Compatibility” tab/Change settings for all users. Select Windows 7 under the Compatibility mode and tick the "Run this program as an administrator" box. Click “Apply” and then “OK”.
  • Double click on “Setup.exe” to start the installation process. Follow the instructions on the screen during this process. The necessary files for the BioFlux Software will be installed, including third party drivers from National Instruments.
  • Upon completion of the installation of BioFlux Full Install V2_5_0_8, do not restart the You will need to install an updated version of the software. In the BioFlux Control Software Patch v2_6_0_9 file, set the software to run as an administrator and Windows 7 compatible before running the update. Run the update to install version V2_6_0_9. The Patch is included with the installation software package in the USB drive.
  • Restart the computer
  • From time to time, Fluxion may release updates to the BioFlux Software. To install these updates, run the Setup file from its folder as an administrator and Windows 7 compatible and it will automatically install the update to the main application program.
  • If you are using a smaller monitor, we have found that with a monitor resolution 1280 x 800 on a 12-inch laptop monitor is sufficient to view the entire faceplate of the software.

Hardware Connections

BioFlux Controller

Once the BioFlux Software is installed, you can connect the USB cable into the computer. The software will recognize the Controller automatically, and the red light indicating ‘Simulation’ mode will disappear.

The first time you connect the instrument, you will need to enter that unit’s specific calibration constants into the BioFlux Software.

To do so, open up the ‘System – Calibration Data…’ menu which will bring up the calibration dialog box:

The BioFlux One has Regulator 1 only. Regulator 1 values can be found on the back of the BioFlux Controller. Enter the calibration values in the dialog box (Regulator 1 PSI/V, Regulator 1 Offset. Values are also required by the software for Regulator 2, so use the same values as for Regulator 1). When finished, click Apply and close the dialog box.

Fluid Properties

BioFlux Software provides support for managing the fluid properties of commonly used reagents, media, and compounds you might use. It is important to note that shear stress is dependent on viscosity, which means that you will want to take care to input the correct fluid properties for each experiment.

A number of media solutions are preset into the software and can be called up during any experiment. Additional fluids can be added in the Fluid Properties dialog box, under the System Menu:

To enter a new fluid, select [Custom] fluid and enter its viscosity (cP). Click ‘Save As…’ to name the fluid and click ‘OK’. Click to ‘Save’ to record your changes. This fluid will then be callable as you run experiments in Manual or AutoRun modes.

Manual Mode

Manual Mode allows the user to operate the BioFlux System in a manual configuration. In this mode, controls are provided for shear flow value, shear flow direction, well status, and volume tracking.

To run an experiment in manual mode, follow these steps:

  • Select the type of BioFlux Plate you plan to use (48-well only)
  • Click ‘Select Active Wells…’ dialog box and select the groupings of channel which will be used for the experiment. Note that certain plate formats will have groups of channels tied to the same control valves.
  • Select the fluids which will be used for each section of the plate.
  • Enter a starting value for shear flow in either regulator.
  • Activate wells by clicking directly on them to flow from that well. Click on them again to turn flow from that well off. Click on the opposite well in that channel to reverse the flow direction.

Important: The Columns 1-4 settings will be used for the entire plate. Utilize the plate image to select the active columns. Click on the left column to program "forward" flow and on the right column for "reverse" flow. The lighter shade of green indicates the inlet side and the dark green is the outlet.

Edit AutoRun

Edit AutoRun is the editor used to define precise protocols and sequences of events which take place during an automated experiment. These protocols are first defined in the ‘Edit AutoRun’ module, then called up in the ‘AutoRun’ module when the experiment is ready to be run. ‘Edit AutoRun’ consists of three menu tabs which constitute a complete experiment:

  1. Protocol Setup – protocols are routines which define the change in shear flow value and direction over a given time course
  2. Sequence Setup – sequences are a series of protocols (imaging and flow) which are assigned to a region(s) of a particular BioFlux Plate.

Protocol Setup

To create protocols, first select the plate you would like to create a protocol for (protocols are not transferable between well plate formats). Protocols consist of a series of intervals, each interval containing a duration, shear value, flow direction, and source (multi-inlet plates only).

  • To create an interval, enter the desired values in the ‘Interval Settings’ box, and click ‘Add’. You can add as many intervals as needed, using the ‘Add’ or ‘Insert’ buttons.
  • You can change an interval by selecting it from the table, changing the settings, and then clicking the ‘Update’ button.
  • The ‘Remove’ button will remove an interval from the protocol. Once the protocol is completed, click ‘Save As’ to create the file.

Sequence Setup

To create sequences, first select the plates you are defining a sequence for (as with protocols, sequences are not transferable between plate formats since the channel layouts are different).

Click on ‘Add Step’ to create the first step in a sequence. For that step, define the relevant parameters: 

  • Step iterations – how many times a given protocol will run during that step (useful for when you want a particular protocol to run many times without having to type in values repetitively into a protocol)
  • Channel Protocols – selects the protocols which will be applied to the flow channels. Plates are divided into groups of channels, each of which can be turned off or on depending on which regions of the plate are being used during a given sequence.

    Important: The flow for channels 1-12 will be applied to the entire plate. To run a portion of the plate, seal the wells that will not be active. 
  • When using only the channels in Columns 5-8 (i.e. channels 13 to 24), at least one of the "enable" box for channels 1-6 or 7-12 must be selected/ticked in order for the flow to apply to the channels in Columns 5-8)
  • The same protocol applying to the channels in Columns 5-8 must be selected and applied to the channels in Columns 1-4 (channels 1-6, or 7-12)
  • When finished editing the step, click ‘Apply’ to reflect the changes. You can add, remove, or edit sequence steps as necessary to complete the desired sequence.
  • When finished, save the sequence, using ‘Save’ or ‘Save As…’ as appropriate.

AutoRun Mode

AutoRun Mode calls up and runs sequences which were previously saved in the Edit AutoRun Mode. To begin an automated sequence, first select the type of plate you will be using from the drop-down menu. Select the active wells using the dialog box. Note that certain plate formats will have groups of channels tied to the same control valves.

Use the ‘View Well Volumes’ dialog box to set and view well volumes. This can be useful when tracking well volumes over longer experiments which may require refilling of the wells when they run out of reagent. Note that this tracking system is based on calculated flow rates and does not have a sensor to detect well volumes. Care should be taken to monitor the wells as they get close to empty.

Select the sequence which will be run. Note that only the sequences created for the selected plate format will be visible from the pull-down menu. Select the fluids being used from the pull-down menus. Use the Start button to initiate the sequence, and the Pause/Abort buttons as needed. When the sequence is completed, you can use the ‘Save Log’ button to save the sequence details in a CSV file (readable by Microsoft Excel or similar application).

Running Experiments

A general outline is provided for typical BioFlux experiments. More detailed assay protocols and methods can be found on support.fluxionbio.com. For further support, please contact Fluxion Biosciences (support@fluxionbio.com).

Cell and reagent preparation

Cells and reagents can generally be prepared using standard lab techniques. It is important to adjust the cell density to an amount appropriate for a microfluidic flow channel. Common values range from 5x10^6 cells/mL for mammalian cells to 10^9 cells/mL for microbial cells. Reagents should typically be pre-warmed to the same temperature at which the experiment will be run.

Cells and reagents can be added to the plate using syringes, pipettes, or liquid handling systems for multi-well plates. When adding reagents, care should be taken to place the tip of the pipette in close proximity to the bottom of the central punch-out region of the plate. Doing so reduces the chance of air bubbles getting caught in the channels.

Connecting the interface

The BioFlux pressure interface with its silicone gasket sits on top of the BioFlux Plates and creates a seal around each individual well. Use the supplied torque driver to tighten down the retaining bolts into the inserts found on the BioFlux Plates. Rotate the driver until you hear a clicking sound, indicated correct torque has been reached.

Caution: Only use the provided torque wrench to tighten down the Interface. Using an Allan key may lead to over tightening and plate malfunction.

Setting up Optional Temperature Control

The BioFlux Heater Plate can be set to temperatures from ambient to 50°C. The temperature controller is found on the front panel of the BioFlux Controller. The main display always reads the current temperature. To change the set point, hold down the ‘star’ key and use the up and down arrows to adjust the set point. When the ‘star’ key is released, the display will return to reading the current temperature.

BioFlux Plates can be placed directly on the Heater Plate to keep them at the specified temperature:

Caution: do not set the heater beyond 50°C. Doing so may cause damage to the heater plate.

Plate preparation

Unless an entire plate will be use at once in an experiment, we recommend sealing the plate with the supplied sealing film upon opening a new plate. Carefully plan out your experiment ahead of time.

Cut out the sealing film with a razor blade only for those wells which are intended be used in the experiment. Reseal all previously used wells before proceeding to running new experiments using new wells on the same plate. See Tips and Tricks

Priming the channels

Before using the BioFlux Plates, it is important to prime the channels which will be used in your experiment. Priming is simply a step to introduce fluid all the way through the channel and serves as a way to prevent air bubbles from getting into the system. Priming or coating of channels is done from the outlet side to the inlet side on the BioFlux low shear (0-20 dyn/cm2) plates, and from the inlet to the outlet side on the BioFlux high shear (0-200 dyn/cm2) plates, owing to the difference in placement of the flow-resistant "serpentine" zones. See below for priming and coating details for each plate type. 

Coating the channel with substrate molecules

You may apply this priming protocol as a coating protocol. For coating plates, use the coating solution instead of PBS. No need to prime and then coat the plate. See section 4.6 below.

To prime or coat a low-shear (0-20 dyn) bcxx 48-well plate follow these steps below:

  • Load at least 40 µL of PBS (or substrate solution) to the outlet well
  • Place the BioFlux interface on the plate and ensure a snug/tight seal
  • Perfuse from outlet to inlet (if using AutoRun, set as reverse) at 20 dyn/cm2 for 40 seconds
  • Direction of flow: outlet to inlet

To prime or coat high-shear (0-200 dyn) caxx 48-well plate follow these steps below:

  • Load at least 40 µL of PBS to the inlet well
  • Place the BioFlux interface on the plate and ensure a snug/tight seal
  • Perfuse from inlet to outlet (if using AutoRun, set as forward) at 150 dyn/cm2 for 6 seconds
  • Direction of flow: inlet to outlet

Coating the channels

BioFlux Plate channels can be coated with a variety of reagents for the purposes of promoting cell adhesion, or to induce an interaction between the coating and subsequent introduction of cells. The coating reagent can be introduced into the outlet well(s) and flowed through the channel much like the priming step. Once the channel is coated, you can follow the recommended protocols or manufacturer’s guidelines for adherence. Often there is a recommended waiting time for the coating to adhere (i.e. 1 hour, 24 hours) as well as a recommended temperature. If the coating calls for a waiting period at physiological temperature, it is acceptable to place the BioFlux Plate in an incubator.

Introducing cells and creating cellular monolayers

Cells can be introduced directly into the input wells. It is important to determine the optimal starting density of cells to ensure that there will be enough cells in the viewing window for observation. Once cells have flowed into the viewing window, they can be left to settle and adhere if a cellular layer is required for the experiment. Note that different cell types may require different coatings to create optimal adhesion to the glass cover slip bottom of the BioFlux Plate. Apply these coatings as described above.

Establishing shear flow

Once the input wells have been filled with reagent (i.e. cell media, compound, etc.), you can connect the Interface and begin running manual or automated shear flow routines using the BioFlux Software (See Section 3 for software control).

A note about flow patterns in the viewing window, i.e. where should you observe the experiment for most uniform flow conditions? The BioFlux microfluidic channels were designed to contain a region of uniform laminar flow within the ‘viewing window’ (see below, Region of laminar flow). Outside of this region, the channel geometry contributes to a region of non-uniform shear and/or higher shear. Therefore, when comparing data from channel to channel in is important to utilize the area denoted below.

BioFlux Plate management and refilling

The entire set of channels on a BioFlux Plate does not need to be used during the same experiment. One or a portion of the channels can be used while preserving the integrity of the remaining channels on the plate. In cases where sterility is a concern, it is recommended to cover the well plate with a sealing film and only punch the wells which will be used in a given experiment.

For longer-term experiments, it is possible to refill the wells when they become close to empty. Note that you should not let the wells run empty while applying shear flow, as this may cause air bubbles to form within the channels.

Image Analysis

Analysis Mode

The Analysis Mode is a suite of image analysis tools used to characterize and quantitate images. Images acquired from your microscope camera can be analyzed using a few commonly used protocols. Note that some information may need to be entered manually for all features to work, for example the frame rate of acquisition. We recommend using the TIFF (.tif) image format.

To load images – Click the ‘Browse’ button to locate your image files on your computer. To open a single image, select that image and click ‘Open’. To load a series of images (such as a time lapse recording), open the directory where the images are saved and click ‘Current Folder’. All supported image types stored in that folder will be loaded onto the Analysis module.

Image Analysis Tools

Calibration – Use this dialog box prior to running any analysis modules that require measurements of distance (i.e. cell diameters, rolling velocity, etc.). Select a feature of known size, typically the BioFlux Plate channels or particles (beads, etc.) of known diameter and calibrated size. You can also stage a micrometer for this purpose.

Draw a line across this feature and set the size to the actual length of this line segment (in units of microns). Click ‘Apply’ to use this as the new calibration scale. Calibration files can be saved and recalled later. This feature is useful when you calibrate each microscope objective that is used on a particular microscope at the beginning of any series of experiments (or when you first start using the BioFlux system). All subsequent analyses can then be run using a saved calibration file rather than re-calibrating between each analysis.

Set Time – Use this dialog box prior to running any analysis that looks at time lapse recordings. Manually input the frame rate (expressed in time between images).

Save As / Build AVI – Use these controls to save images as single images or built into an AVI video file. Single images can be saved with or without measurement overlays.

Analysis Module

Manual – This module provides a series of basic measuring tools to analyze images. The line tool can measure distances between points. The pointer tool (looks like a crosshatch) can identify points for cell counts. Use the CTRL key to select multiple regions or points.

Cell Tracking – This module enables quantitative measurements for movement of cells or cellular projections over time. It can be used for a wide variety of kinetic application, such as: measuring rolling velocity, neuronal projection kinetics, and cell migration velocity. To begin, load a stack of images which were recorded as a time-lapse series (i.e. same field of view). Make sure the Calibration and Set Time dialog boxes have been set correctly (Cell Tracking module is dependent on both of these parameters). Click on ‘Add Track’ and note that the cursor changes to a cross- hatch. Select a cell or point of interest that serves as a starting point and click on that point of the image. Once clicked, the frame will automatically advance to the next frame. Keep clicking on the cell or point of interest until you have captured enough of the motion, then double-click on the last point to end the track. You can add as many tracks as you need to complete the data set. The results can be exported to a data file by using the report generator.

Intensity – This module calculates intensity (brightfield or fluorescence) over an image or region of interest. It can be applied to a single image or a stack of images. Load the image(s) for analysis and use the Threshold tool to select the background region (cursor will turn to eyedropper tool once you click the ‘Threshold’ button). Select either Total (value is all pixels above threshold summed) or Mean (the mean intensity of all pixels above threshold). Select the range of images you want to analyze. You can analyze a single image, an entire stack, or a portion of a stack (provided they are all in sequential order, such as frames 10-20). You can also select a region of interest using the rectangle tool, which is helpful if you want to exclude boundary areas or focus on a particular cell or region.

Image Stacker – This module overlays images on top of each other to project how a particular object has traveled over time. It can be used to quantitate rolling velocity and other kinetic measurements. Load a stack of images and ensure that the proper calibration and timing is entered. Choose the imaging mode (brightfield or fluorescence) and a range of frames to stack, then click ‘Create’. The resulting stacked image can them be analyzed for velocity of cells or other points of interest by drawing a line across the starting and ending point of the “streaked” image:

Area Coverage – This module calculates the area coverage (brightfield or fluorescence) over an image or region of interest. It can be applied to a single image or a stack of images. Load the image(s) for analysis. Select a region of interest using the rectangle tool, which is helpful if you want to exclude boundary areas or focus on a particular cell or region. Use the Threshold tool to subtract the background. Tick the ‘Highlight Threshold Pixels” box to show the highlighted areas. In this example, the area covered in a region of interest from a stack of images is calculated using the inclusive threshold type.

Cleaning, Sterilization, and Maintenance

To decontaminate the system after routine use

This applies to experimental runs where there is no overt contamination (e.g. no visible blood on the interface).

BioFlux Interface

After you have completed the experiments, inspect the interface carefully. If you do not see any liquid inside the interface – you can saturate a paper towel with 70% isopropanol or ethanol and wipe the interface thoroughly. Decontamination time with 70% alcohol is 30 minutes of wet exposure to the biocide. Allow to air dry following decontamination.

BioFlux Vapor Trap

The Vapor Trap can be cleaned with a paper towel saturated with 70% isopropanol or ethanol. Decontamination time with 70% alcohol is 30 minutes of wet exposure to the biocide. Allow to air dry following decontamination.

BioFlux Plates

BioFlux plates are shipped sterile and do not require cleaning. Do not autoclave.

BioFlux Tubing

For routine cleaning, tubing can be wiped with 70% isopropanol or ethanol on the external surface.

To decontaminate the system after overt contamination

This applies to situations where blood or other liquids are visible inside the interface, tubing and/or vapor trap.

  • Immediately place the interface still connected to the system and the BioFlux plate on an absorbent mat or plastic-backed paper lab mat.
  • Remove the tubing from the controller side of the vapor trap leaving the filters attached to the vapor trap.
  • Move entire assembly, vapor trap included, and the paper mat to a Biosafety cabinet.
  • Wear the appropriate personal protective equipment. Inspect the equipment for sites of contamination – start at the Interface and follow the contamination through the tubing.
  • Wrap Interface for autoclaving and sterilize on-site in a certified autoclave. Recommended steam sterilization (autoclave) settings are 121˚C for a maximum of 60 minutes.
  • Following sterilization, if necessary, the interface can be cleaned with lab detergent (Alconox or equivalent) and rinsed with distilled water.
  • If there is contamination in the tubing, dispose of it as Biohazard waste. Do NOT attempt to clean the tubing as it will be difficult to assure sterility for subsequent experiments. Tubing cannot be autoclaved.
  • If the vapor trap is contaminated, it needs to be autoclaved as well. To prepare it for autoclaving, remove all tubing and filters (dispose of overtly-contaminated parts), loosen all screws, and wrap for autoclaving. Recommended steam sterilization (autoclave) settings are 121˚C for a maximum of 60 minutes. Following the autoclaving, wash the inside of the vapor trap using laboratory approved detergent. Dry completely and reassemble.

Replacement parts and preventative maintenance

It may be necessary to periodically order replacement parts and increase the frequency of preventive maintenance. The parts which may need to be replaced are available from Fluxion or an authorized regional representative, and include: tubing sets, filters, tubing connectors, interface gaskets and vapor trap inserts. 

Replacement Component

Fluxion Order Number

Tubing harness

200-0032

Gasket, 24-well Interface

800-0068

Gasket, 48-well Interface

800-0069

0.2uM replacement filters, Qty 16

910-0017

Troubleshooting Guide

Troubleshooting Hints

Problem Observed

Troubleshooting Recommendation

Controller

 

LED lights on front panel are not coming on when power switch is turned on

Check that the power supply and electrical cord are properly connected and plugged into an appropriate power outlet

Compressor is running continuously

Check all pneumatic connections to ensure that each tube is properly inserted into the push connectors. Check that the tubing manifold is properly tightened down by the two securing bolts. Check the plate orientation.

Temperature controller is displaying ‘Input Fail’

Make sure the Heating Plate is properly connected to the back panel

Interface

 

Air seems to be hissing out of the plate when the Interface is connected.

Check that all the retaining screws have been properly tightened down on to the BioFlux Plate using the provided torque wrench. Check that each of the tubes is fully inserted into the push connectors on the Interface.

Heating Plate

 

Heating Plate is not heating up.

Check that the Heater Plate cable is properly connected into the back panel. Check that the temperature controller has been set to the desired temperature.

BioFlux Plates

 

Reagents do not appear to be flowing through the channels.

Check first that the Interface is applying a pressure. You can check this by removing the Interface from the plate and turning the shear up to a high level. You should feel air pressure coming out of the wells.

If air pressure is coming out, then inspect the channels for any visible air bubbles. Try removing the fluidic contents and re-priming with fluid, making sure to pipette the fluids into the lower punch-outs on the BioFlux Plate wells.

Software

 

Software doesn’t seem to be controlling the Controller. Red light is indicating that you are in ‘Simulation’ mode.

Check that the USB cable is connected between the Controller and the computer. If it is, try unplugging the cable, wait a few seconds, and then plug it back in. If still no connection, go to System Menu – Data Acquisition Setup and see if there is a device recognized in the menu. If no device is recognized, check that the USB port is 2.0.

 

Warranty Information 

Fluxion Biosciences warrants BioFlux instruments against defects in materials and workmanship for a period of one year from date of installation. During the warranty period, Fluxion will, at its option, repair or replace products that prove to be defective. Fluxion warrants that its software and firmware designed for use with a CPU will execute its programming instructions when properly installed on that CPU. Fluxion does not warrant that the operation of the CPU, or software, or firmware will be uninterrupted or error-free.

Warranty service

Warranty service is performed at no charge and at Fluxion's option in one of the following methods: 

  • A service representative is dispatched to the customer’s facility.
  • The product is returned and repaired at a Fluxion repair facility.
  • Replacement parts with appropriate installation instructions are sent to the customer. 

The foregoing warranty shall not apply to defects resulting from:

  • Improper or inadequate maintenance, adjustment, calibration
  • Buyer-supplied software, hardware, or consumables
  • Unauthorized modification or misuse
  • Operation outside of the environmental and electrical specifications for the system
  • Improper site preparation

THE WARRANTY SET FORTH IS EXCLUSIVE AND NO OTHER WARRANTY, WHETHER WRITTEN OR ORAL, IS EXPRESSED OR IMPLIED. FLUXION SPECIFICALLY DISCLAIMS THE IMPLIED WARRANTIES OF MERCHANTABILITY AND FITNESS FOR A PARTICULAR PURPOSE.

Limitation of Remedies and Liability

THE REMEDIES PROVIDED HEREIN ARE BUYER’S SOLE AND EXCLUSIVE REMEDIES. IN NO EVENT SHALL FLUXION BE LIABLE FOR DIRECT, INDIRECT, SPECIAL, INCIDENTAL, OR CONSEQUENTIAL DAMAGES (INCLUDING LOSS OF PROFITS) WHETHER BASED ON CONTRACT, TORT OR ANY OTHER LEGAL THEORY.

Tips and Tricks 

Instrument control:

Disable live updates on PC which may restart the computer controlling the BioFlux system. 

Also configure power saving settings so that the computer will not shut down or hibernate during BioFlux operation- especially important for overnight experiments. Your best bet is to disable any connection to the internet or intranet.

Plate sealing film:

If you see a gap along the perimeter after you place the film on the plate, lab tape placed along the outside edge (long side) of the plate over the film will seal it better than film alone.

When you punch the plate with a needle, try to center the punch inside the well. You only need to punch the wells that you are working with.

It is easier to peel the film back than to punch through it for liquid changes.

Imaging:

Always image the most unstable fluorophore first. For example, in a sample dual-stained with an Alexa dye and a traditional fluorophore e.g. FITC, it is better to image the FITC first because it photo-bleaches easily compared to the more stable fluorophore. This is especially important for dyes in the DAPI range, the high intensity wavelength of light will visibly bleach regions of interest.

High resolution fluorescent images can be obtained with objectives that are not “phase” or “PH” specified–i.e. those with a higher numerical aperture, closer working distance, and an unrestricted optical path because more light can be transmitted back to the camera / oculars leading to brighter fluorescence and better resolution.

Plates:

When you handle the plates, try not to apply pressure to the coverslip glass on the bottom – it can be cracked easily in this way.

When priming channels of interest, it is important to inspect channels after priming to make sure that the entire path has been filled with liquid. If you see bubbles upon initial priming, you may remove them by ramping up shear to higher levels. If this doesn’t work, it may help to push fluid all the way out of the channels and reprime. Repriming with 50% isopropanol might be of use as it has lower surface tension and fills in the microfluidic path better than water (follow with a water wash). Finally, if this doesn’t work, we have outlined a procedure to expel trapped bubbles by pressurizing both the inlet and outlet wells at the same time. A detailed protocol may be found on our support site at support.fluxionbio.com.

Bubbles:

To avoid bubbles, equilibrate media to the experimental temperature. Avoid temperature swings. If you work in a lab with strong air conditioning for instance, it is advisable to warm all equipment and reagents in an incubator to avoid spontaneous bubbles.

Bubble formation may occur with use of the heater plate when there is a major difference between ambient temperature and experimental temperature. In this case, you might want to try insulating the plate, degassing buffers, or using a traditional incubator instead.

Degassing buffers using vacuum filters, vacuum extraction, boiling (autoclaving) or other methods may be useful in avoiding bubbles.

Sometimes a bubble is lurking in the wells of interest or the serpentine from the priming process. Therefore, before starting an experiment, always check around the well-microfluidic interface and in the serpentine for residual bubbles. Deal with them as above (plates). 

The ‘View Well Volumes’ dialog box:

Use the ‘View Well Volumes’ dialog box to set and monitor projected well volumes during experiments. While some customers use this feature, the accuracy of the projected volumes is limited by cases where temperature is not well controlled, and the fluid properties are not well understood. Tracking is based on calculated flow rates and does not have a sensor to detect well volumes. For that reason, for most users, we do not recommend using this feature or relying on projected well volumes.

End User License Agreement

By opening and using the BioFlux Software Product, (1) you acknowledge that you have read, understand, and agree to be bound by this Agreement, and (2) you represent that you have the authority to enter into this Agreement on behalf of End User and to bind End User to the terms of this Agreement.  If you do not agree to all terms and conditions of this Agreement, or if you do not have such authority, you should return the BioFlux Software Product in accordance with Fluxion’s then-current return procedures.

  1. The FLUXION Software Product is supplied to End User as an executable file that can be installed on computers for use with the BioFlux instrumentation.
  2. Use of the FLUXION Software Product is limited to the End User’s internal business purposes.
  3. Title to and ownership of the FLUXION Software Product shall remain with FLUXION and its suppliers.
  4. The End User may not (a) alter or modify the FLUXION Software Product; (b) reverse engineer, decompile, disassemble, or in any way attempt to derive the source code for the FLUXION Software Product; or (c) transfer the FLUXION Software Product to any third party or make the FLUXION Software Product available to any third party as part of any time-sharing or service bureau arrangement.
  5. The End User may make a reasonable number of copies of the FLUXION Software Product for its internal business use, provided that the End User reproduces all copyright and other proprietary rights notices included on or within the Software as delivered to the End User.
  6. The End User will not export or re-export the FLUXION Software Product without complying with the relevant United States or foreign government laws and regulations.
  7. ALL EXPRESS AND IMPLIED WARRANTIES AND CONDITIONS, including but not limited to the implied warranties or conditions of merchantability, fitness for a particular purpose, title, and non-infringement of third-party rights REGARDING THE FLUXION SOFTWARE PRODUCT BY FLUXION’S SUPPLIERS, ARE DISCLAIMED.
  8. All consequential, special, and indirect damages are disclaimed on behalf of Fluxion’s suppliers.
  9. To the maximum extent permitted under applicable law, Fluxion is expressly named as an intended third-party beneficiary of the EULA, with the right to enforce its terms directly against the End User.

Declaration of Conformance

 

Fluxion Biosciences

1600 Harbor Bay Parkway, #150
Alameda, CA 94502
(650) 241-4777
www.fluxionbio.com

Product: BioFlux Controller

Model No.: 200

Serial No: <As marked on unit>

The undersigned hereby declares, on behalf of Fluxion Biosciences, Inc. of Alameda, CA, that the above-referenced product, to which this declaration relates, is in conformity with the provisions of:

Requirement:      Standard:    Document Identification:
EMC  EN 55022: 1998 + A1: 2000 + A2: 2003 Class A R0806251-1
  EN 61326-1: 2006 R0806251-326
SAFETY EN 61010: 2001  R0806251-3

The Technical Construction File required by this Directive is maintained at the corporate headquarters of Fluxion Biosciences, Inc, 1600 Harbor Bay Parkway, Alameda, CA.

The product herewith complies with the above-mentioned provisions of normative European Council Directives and carries the CE marking accordingly.

Signature on file

Jeff Jensen

CEO

 

Quick Reference Sheet

 

BIOFLUX PLATE FLUIDIC LAYOUTS

 

24-well Microplate

 

48-well Microplate