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Chapter 3: Using the

Amnis

®

FlowSight

®

Imaging Flow Cytometer

Operating the Amnis

®

FlowSight

®

Imaging Flow Cytometer

This chapter describes the operation of the FlowSight

®

System using the INSPIRE

™

for FlowSight Software. Daily oper-

ation involves an initialization of the system and calibration using the Calibration bead reagent, followed by sample runs
and data acquisition, and finally a shutdown procedure which sterilizes the system and prepares for the instrument for
use the following day. Optimizing instrument setup for sample runs is also described in this chapter.

l

User Interface

l

Reagents

l

Daily Calibration and Testing

l

Instrument Setup

l

Data Acquisition

l

Daily Shutdown

l

AutoSampler

l

Quantitative Imaging

For Research Use Only. Not for use in diagnostic procedures.

8

Amnis

®

FlowSight

®

Imaging Flow Cytometer User Manual

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Summary of Contents for Amnis FlowSight

Page 1: ...diagnostic procedures 780 01286 03 Rev D February 2020 Technical Support Telephone 512 381 4397 North America Toll Free 1 877 785 2323 International Toll Free 800 2939 4959 Email support luminexcorp com www luminexcorp com Luminex Corporation 12212 Technology Blvd Austin Texas 78727 U S A ...

Page 2: ...the Amnis FlowSight Imaging Flow Cytometer 8 Reagents 15 Daily Startup and Calibration 16 Data Acquisition 19 Setting up the Work Area 27 Quick Start Guide to FlowSight System Operation 30 AutoSampler option 32 Menus 39 Chapter 4 Troubleshooting 42 For Research Use Only Not for use in diagnostic procedures ii ...

Page 3: ...ergent Using gloves clean the sample portal and sample elevator with a 10 bleach solution Dispose of waste using proper precautions and in accordance with local regulations l Preventative maintenance The FlowSight System contains no serviceable parts Only Luminex trained tech nicians are allowed to align the laser beams or otherwise repair or maintain the instrument The instrument flu idic system ...

Page 4: ...Interior side panels near release mechanisms and next to hood interlocks Risk of exposure to hazardous laser radiation On the back of the instrument No laser radiation is accessible to the user during normal instrument operation Electrical Safety Equipment ratings The FlowSight System is rated to the following specifications 100 240 VAC 50 60 Hz and 1 5 A For Research Use Only Not for use in diagn...

Page 5: ...he user during normal instrument operation When the hood is removed interlocks on the instrument turn the lasers off The FlowSight System may have the following lasers Wavelength Maximum Power 400 413 nm 150 mW 483 493 nm 60 mW 558 562 nm 50 mW 635 647 nm 150 mW 775 795 nm 90 mW The following laser warning label appears on the interior side panels near release mechanisms Using controls making adju...

Page 6: ...he sample to inactivating conditions It is recommended that samples be fixed in 2 paraformaldehyde for at least 10 minutes before running the samples on the FlowSight System The use containment and disposal of biologically hazardous materials are required to be in accordance with Personnel Protective Equipment Directive 93 95 E and are the responsibility of the end user Follow all local state and ...

Page 7: ... la responsabilité de l utilisateur Respectez la réglementation en vigueur pour le traitement et l élimination des déchets dans des réservoirs prévus à cet effet Prévenir l accumulation des déchets en vidant le réservoir lorsque l indicateur indique qu il est plein Stériliser les ins truments de routine apre chaque journée d utilisation Notez que cette procédure ne garantit pas la stérilité vis à ...

Page 8: ...ation Have a sample of cells each labeled with a single color for each fluorochrome used i e FITC only cells PE only cells etc l Cell Aggregation Minimize aggregation problems by straining the sample through a 70 micron nylon mesh strainer or by using an anti clumping buffer such as EDTA or Accumax prior to fixation l Fixation If fixation is desired thoroughly fix cells with 1 formalin on ice for ...

Page 9: ...yes are AF488 PE PE TxRed PE Cy5 SSC Ch6 2 laser 488 642 system Ideal dyes are AF488 PE PE TxRed SSC Ch6 and AF647 APC Cy7 3 laser 405 488 642 system Ideal dyes are AF488 PE PE TxRed SSC Ch6 and DAPI AF647 APC Cy7 Band pass filter values assume 3 laser configuration For Research Use Only Not for use in diagnostic procedures 7 Amnis FlowSight Imaging Flow Cytometer User Manual ...

Page 10: ...d reagent followed by sample runs and data acquisition and finally a shutdown procedure which sterilizes the system and prepares for the instrument for use the following day Optimizing instrument setup for sample runs is also described in this chapter l User Interface l Reagents l Daily Calibration and Testing l Instrument Setup l Data Acquisition l Daily Shutdown l AutoSampler l Quantitative Imag...

Page 11: ...tion is displayed along the bottom of the window The Image Gallery Images are displayed in the image gallery during setup and acquisition Image Gallery Tools Icon Name Description All Select the population to view Pause Pause Resume the display Up Down Move up or down in the image gallery while paused Zoom in Enlarges the imagery Zoom out Resets the zoom For Research Use Only Not for use in diagno...

Page 12: ...gram Create a histogram tool Scatter Plot Create a bivariate scatter plot tool Pointer Reset cursor to pointer Line region Draw a line region on a histogram Rectangle region Draw a rectangular region on a scatterplot Oval region Draw an oval region on a scatterplot Polygon region Draw a polygon region on a scatterplot Select All Selects all plots in analysis area Tile Tiles the graphs in the analy...

Page 13: ... Panel The instrument control panel provides tools to control instrument operation data acquisition and status For Research Use Only Not for use in diagnostic procedures 11 Amnis FlowSight Imaging Flow Cytometer User Manual ...

Page 14: ...er type the num ber of events and choose the population to collect Begin Acquisition Custom Filename Text Type the filename Seq Choose the beginning sequence number Navigate to the folder to save the data Count Enter the number of events to collect of Choose the population to collect Collect a different population while counting the first population Click on the bracket to break the link and selec...

Page 15: ...rently set to OFF at 10 mW of power Brightfield illumination is shown as ON in channels 1 and 9 Sets the Intensity of the brightfield to 800 counts Adjust the speed and sensitivity for the run Run fluidics Stop fluidics Speed and Sensitivity are inversely related Focus and Centering can be adjusted using the right and left arrows Runs the focus pan test and sets focus automatically Runs the startu...

Page 16: ...mpensation is being applied to the Intensity feature and images Yellow calibrations and tests not run Red one or more calibrations or tests failed Green all calibrations and tests have passed For Research Use Only Not for use in diagnostic procedures 14 Amnis FlowSight Imaging Flow Cytometer User Manual ...

Page 17: ...nk It is recommended that the waste bottle contain 10 bleach when full Sheath Fluid Two bottles are provided one labeled Sheath to be filled with phosphate buffered saline PBS with no surfactants for running samples and one labeled Rinse to be filled with de ionized DI water for rinsing the instrument during shut down Fluid is drawn from these bottles into the sheath and flush syringe pumps The sh...

Page 18: ...o ON 2 Log on with the user name Amnis and password IS100 3 Launch the FlowSight Software using the shortcut on the desktop after the computers and instrument have started Preparing to Run Calibration 1 Fill the rinse bottle with Milli Q deionized water and the sheath bottle with PBS 2 Empty the waste tank Push on the quick disconnect buttons to remove the tubing from the waste tank Add 200 mL of ...

Page 19: ...ed under the Instrument menu NOTE If adjustments are made to the instrument in order to pass ASSIST repeat all calibrations and tests to save the new settings to the database Reporting the ASSIST Data All calibration and test results are saved to a database and can be accessed for reporting and tracking instrument per formance l To see the result of the last time a calibration or test was run clic...

Page 20: ...For Research Use Only Not for use in diagnostic procedures 18 Amnis FlowSight Imaging Flow Cytometer User Manual ...

Page 21: ...ith the brightest stains to set the sensitivity for the run l Single color DNA dye control NO BF or SSC to ensure correct dye concentration l 10 bleach to wash out DNA dye followed by PBS l Single color fluorescence compensation samples no DNA dye NO BF or SSC all channels enabled l The rest of the experimental samples with DNA dye if applicable Loading and Running the Sample 1 Choose Load Default...

Page 22: ...ult saturation color on the images is cyan but can be changed in the display settings section In the examples below the objects are not saturating in channels 2 5 or 7 but saturating in channel 11 indicated by the events lining up at 4x103 counts 4 Select Brightfield channels Default is Channels 1 and 9 Click Set Intensity 5 Set the display properties if needed see Setting the Image Display Proper...

Page 23: ...l Area The number of pixels in an image reported in square microns l Max Gradient Intensity The value of the largest slope spanning three pixels in an image This feature measures image contrast or focus quality l Intensity The integrated intensity of the entire object image the sum of all pixel intensities in an image background subtracted l Mean Pixel The average pixel intensity in an image backg...

Page 24: ...n the Sequence box is appended to the file name followed by the rif extension The sequence num ber increases by 1 with each successive data acquisition Files collected with BF off will be appended with noBF File names must be 256 or fewer characters in length including the path and file extension In addition file names can not contain the following characters or 3 Optional Choose fcs file from the...

Page 25: ...y be collected manually by turning bright field and the SSC laser OFF and enabling all channels In either case the experimental data files may be compensated in IDEAS Software during analysis Collect compensation files with the same laser power settings as the experimental samples 1 To use the compensation wizard click to open the wizard list and select Compensation 2 Click Load Compensation Sampl...

Page 26: ...all are positive 7 Click Acquire to save the file The compensation coefficients are calculated and added as shown in the table Compensation is applied to the Intensity feature and images Two scatter plots are added to the work area One of Uncompensated Intensity and one of Intensity for the peak channel vs the adjacent channel is added to the work area For Research Use Only Not for use in diagnost...

Page 27: ...he 2x2 submatrix from 0 189 to 0 10 changes the compensation as shown from the graph above properly compensated to the graph below under compensated 10 Repeat Load Compensation Sample step for each single color compensation sample in the experiment For Research Use Only Not for use in diagnostic procedures 25 Amnis FlowSight Imaging Flow Cytometer User Manual ...

Page 28: ...t the display as the images are being collected The display can also be set manually by the following procedure NOTE These settings adjust the monitor display and do not change the pixel intensities of the images collected Using the Wizard 1 Click on the wand next to the analysis area tools and select the Display Settings Wizard 2 Select the channels to set and click Finish The wizard will adjust ...

Page 29: ...oise from the image Click dragging the vertical green line on the right side allows you to set the display pixel intensity to 255 for all intensities that appear to the right of that line If a non linear mapping occurs by moving the yellow cross hair click Set Linear Curve to return to a linear trans formation NOTE Changing the display properties does not change the pixel intensity data They are f...

Page 30: ...The sequence of objects Raw Centroid X none The central tendency of the pixels along the X Axis and Y Axis respect ively Raw Centroid Y none The central tendency of the pixels along the X Axis and Y Axis respect ively Raw Max Pixel MC_Ch01 through MC_Ch12 The largest pixel value Raw Min Pixel MC_Ch01 through MC_Ch12 The lowest pixel value Uncompensated_ Intensity MC_Ch01 through MC_Ch12 The sum of...

Page 31: ...y viewing the populations created by gates in the image gallery 5 Create scatterplots and histograms as required For Research Use Only Not for use in diagnostic procedures 29 Amnis FlowSight Imaging Flow Cytometer User Manual ...

Page 32: ...tion on graphing tools moving regions or panels or managing populations Quick Start Guide to FlowSight System Operation 1 Power up the system and launch the FlowSight System application For Research Use Only Not for use in diagnostic procedures 30 Amnis FlowSight Imaging Flow Cytometer User Manual ...

Page 33: ...lots and regions to identify the cells to collect or collect All events 11 Set the acquisition parameters including file name destination folder number of events and population s to col lect 12 Choose file format either rif IDEAS fcs FACS software or both from File menu 13 Compensate data if needed Data can be recompensed in IDEAS Software post acquisition a Open the compensation wizard b Load a c...

Page 34: ...and calibrate as described under normal operations 2 Create Instrument Settings Template s ist to be used for each well on your plate To do this run an exper imental sample manually with all of the fluorescence dyes to be used in the experiment see INSPIRE Setup Quick Start Guide Save each relevant template 3 Create a Well Plate Definition def that assigns instrument settings to wells names to the...

Page 35: ... the delete key 4 Click OK when done 5 Browse for a previously saved definition to edit by clicking on the folder icon or continue to create a new definition 6 Name the plate definition 7 At a minimum each well requires an Output File Path Max Acquisition Time and Template File in order to be con sidered defined Optional parameters can be added to the definition in the next step NOTE Luminex highl...

Page 36: ...ters To delete a cus tom parameter select it and use the delete key Click OK when done 12 Select wells to define by clicking a individually or Ctrl click shift click for multi select b rows or columns c color d the Select Defined button or e All NOTE Selecting and defining wells with shared parameters first and then refining the definition for sets of wells makes it easier to organize the definiti...

Page 37: ...lue in the box corresponding to that well Below is an example of a Well Plate Definition using several para meters and showing only defined wells 14 Highly recommended select Error notification Email from the list of Standard parameters and type in the user email address in the Apply to Selected box 15 When done click Save NOTE A warning may be displayed if there are undefined or partially defined...

Page 38: ...the cur rent sample is finished 3 Select the wells to run they will appear in the list Load the Plate 1 Click Eject Tray to extend the plate nest 2 Add at a minimum 75 μL samples to the wells of a u bottom 96 well plate and cover with Sigma Aldrich X Pierce Film XP 100 Cat 2722502 and load the plate into the AutoSampler 3 Place your plate in the nest with well A1 positioned at the upper left corne...

Page 39: ...error occurs on three consecutive wells the AutoSampler aborts the plate and sterilizes the instru ment if the Sterilize after running plate box is checked Report l A well plate report txt file will be saved to the folder designated in the Output File Path of the plate definition at the end of the run either when it was stopped manually or completed the entire plate l If batching was included in t...

Page 40: ...t is close enough to proceed and this requires Brightfield if the template has BF off then BF must be turned on This step occurs whether validation is ON or OFF in the plate definition NOTE Luminex highly recommends that the validation is Yes in the plate definition for every well l When the validation is Yes for the well the object rate and synchrony is then tested and must pass some pre determin...

Page 41: ...ttings as a template for future use Template file names are appended with the suffix ist They are saved in the INSPIRE Data folder l Load Default Template Loads factory settings l Generate RIF file Check to save a Raw Image File during acquisition l Generate FCS file Check to save a Flow Cytometry Standard file during acquisition l Exit and Shutdown Instrument Turns off the instrument control syst...

Page 42: ...mple Load Line Cleans out the sample load line l View Tank Levels Opens the fluid level window l Service Scripts For field service personnel only l Usage Monitor is available to find the information on scripts that have run l Options l Analysis menu Access the Feature Population and Region Managers Functionality is the same as for IDEAS Software Refer to the IDEAS User Manual for more information ...

Page 43: ...e compensation wizard l Load Matrix Apply an existing compensation matrix l View matrix Opens the compensation matrix view l Edit matrix Opens the compensation matrix for editing l Clear matrix Stops applying a compensation matrix l Advanced menu For field service personnel only l Help Access the current software version number l About Software information including version number l Help Opens the...

Page 44: ... save data Software l see INSPIRE appears to freeze l see INSPIRE fails to launch l see Plots fail to update or update slowly l see Troubleshooting Image l see No Images l see Imaging and acquisition rate is erratic or appears frozen l see Objects appear streaked l see Objects are not centered in the channel l see Objects are rotating in the core stream l see Objects are out of focus l see Objects...

Page 45: ...eath is dPBS De gas the sheath as appropriate Third party sheath buffers cannot be used on the FlowSight System Fluid lines are leaking With the system powered down look for leaking sheath Verify the fluid lines mount snuggly into position Call Luminex Technical Support Fluidics respond sluggishly Air buffer in the sheath syringe is not correct The sheath syringe should contain 2 4 mL of air to bu...

Page 46: ...nation from previous samples DNA dye from pre vious sample is labeling current sample DNA dyes must be thoroughly flushed from the sample lines to prevent resid ual dye from labeling subsequent samples Load a sample of 10 bleach fol lowed by a PBS wash to remove all traces of the DNA dye in the instrument or run the sterilize script 30min Cells from the pre vious sample are appearing in current sa...

Page 47: ...Horizontal Laser Cal ibration failure Verify the laser turns on and can set power properly Completely power down the FlowSight System and power back up to re run the test Verify spa tial offsets passed Retro Calibration fail ure Verify the laser turns on and can set power properly Verify spatial offsets and frame offsets passed Side Scatter Cal ibration failure Verify the 785 SSC laser turns on an...

Page 48: ...compensation control sample has more than 10 pos itive events and are as bright as possible IgG capture beads or a cell line stained with a single fluorochrome may be used for comp controls Cells are stained with more than one fluoro chrome Compensation controls must be a sample with a single fluorochrome label in a single tube Each fluorochrome must be run separately Cell concentration is too low...

Page 49: ... the template For optimal plot update rates limit the number of plots to 15 Parent population has no qualifying events Right click on the plot select graph properties and change the selected population to all or a population that has qualifying events Plots are scaled incor rectly In the plot tool bar press the magnifying glass and rescale the plot Data file fails to collect No events qualify for ...

Page 50: ...5 SSC laser to 40 mW Set the laser powers to maximum and decrease them to prevent pixel saturation Make sure the brightfield lamp is turned on and click Set Intensity The sample is too con centrated The process of object detection can safely handle up to 4000 objects per second The maximum sample concentration is 4 5x10 8 cells per mL with the recommended concentration 1 10 x10 7 cells per mL To d...

Page 51: ...system Autofocus and centering is not tracking properly In the Focus and Centering section adjust focus and centering left or right until the images are centered and in optimal focus The two bright field images are not of the same cell Frame offset is incorrect Run calibration particles on the FlowSight Instrument Load the default template and verify brightfield is in channel 1 and 9 at 800 counts...

Page 52: ...er power to prevent pixel saturation Saturation is indicated in the image gallery by pixels colored in a contrasting color generally red or white Set the brightfield intensity to 800 counts by pressing Set Intensity The sheath syringe is empty Load sheath then go to the instrument drop down and run prime There is a clog or air bubble in the system Run the Purge Bubbles script from the instrument d...

Page 53: ... Unstable fluidics Air or clog in system Large variation in brightfield intensity levels Large flow speed vari ation due to air Run the purge bubbles script from the instrument drop down menu See solutions for unstable fluidics Light source delivering variable output Power down and power up the instrument if this does not fix the problem call Luminex Technical Support Brightfield intens ity level ...

Page 54: ...o modify its products and services at any time This guide is subject to change without notice Although prepared to ensure accuracy Luminex assumes no liability for errors or omissions or for any damages resulting from the application or use of this information Amnis FlowSight IDEAS ImageStream and Luminex are trademarks of Luminex Corporation registered in the U S and other countries INSPIRE is a ...

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