FloMax Software for Cytometry. Operating Manual Instrument Control and Acquisition. FloMax Operating Manual Instrument Control and Acquisition

Transcription

1 FloMax Software for Cytometry Operating Manual Instrument Control and Acquisition 1

2 Contents Introduction to FloMax Software Acquisition and Instrument Control...3 Operating Basics - Preparing FloMax for a Measurement...4 Operating Basics - Starting a Measurement...5 Operating Basics - Open the Instrument Settings Box...6 Operating Basics - Loading and Saving Instrument Settings...7 Operating Basics - Selecting Parameters for Analysis...8 Operating Basics - Trigger, Labels, and Others...9 Operating Basics - Gain...10 Operating Basics - Sample Speed...11 Operating Basics - L-L and U-L Thresholds...12 Acquisition Setup - Preset Number of Events and/or Analysis Time...13 Acquisition Setup - Prerun, Stabilizing, and Clean Time...14 Acquisition Setup - Time Parameter...15 Sorting...16 Controlling the Measurement - Start, Pause, Clear, End...17 Controlling the Measurement - Clean, Status Indication...18 Controlling the Measurement - Trigger Selection...19 Controlling the Measurement - Particle Rate, Count, Test Pulses...20 Absolute Cell Counting - Analysing the Particle Concentration...21 Saving the Measurement File...22 Panels - Overview...23 Panels - Definition...24 Panels - Acquisition...25 Panels - Optimizing...26 Life Gating...27 Crosstalk Compensation - Realtime Online Compensation...28 Open a Measurement File

3 Introduction to FloMax Software Acquisition and Instrument Control What... functions are offered by FloMax in conjunction with Partec flow cytometers and sample automates in addition to its analysis functions? Instrument Control and Data Acquisition Realtime Acquisition True Absolute Counting Complete walk-away sample preparation and analysis with Robby sample automat FCS Data Analysis and Report Generation Flexible data display in one- and twoparameter histograms and dotplots Multiparameter Gating with logical gates Colour Crosstalk Compensation DNA Cell Cycle and Peak Analysis Calculated Parameters Panel System Automated Report Generation in conjunction with MS Word and Excel other manuals are required? This manual is a supplement to FloMax Operating Manual Data Analysis. Most of the analysis functions are available during acquisition as well. Please use this manual together with Part I. 3

4 Operating Basics - Preparing FloMax for a Measurement 1. Make sure your flow cytometer is prepared for analysis. Be sure fresh sheath fluid is present and the waste container is empty. Please refer to the Instrument Operating Manual for details about preparing the instrument for analysis. 2. Start FloMax software: Doubleclick on FloMax icon on the Windows desktop. FloMax Icon on the desktop. 3. Select View - Page Layout for a basic page layout., e.g. with 6 plots. 4. Select Acquistition - Instrument Settings to select suited instrument settings. Refer to Panel description how to run a panel series. Refer to Automat description how to operate the automat. Page Layout Selection dialog. 4

5 Operating Basics - Starting a Measurement Make sure the flow cytometer is ready for analysis and FloMax software is prepared for the measurement. s See pp. 6 on how to change instrument settings. If sheath container is empty or waste container is full, an error message is displayed and acquisition cannot be started. When removing a tube from the sample port, the BioSafety system is activated to prevent contamination by hazardeous material*. Simultaneously, a cleaning cycle is performed to avoid cross-contamination between individual samples. or Fill up sample tube with 1-2 ml of the ready prepared sample suspension. The tube should not be filled up more than 2/3 of its volume. Insert sample tube onto the sample port until you recognize a "click". This should go fast. Now the measurement (acquisition) starts automatically - FloMax software indicates the Prerun, Stabilize, Run and Count status. In the Prerun phase, cells quickly transported to the position of analysis, the flow cuvette. During the Stabilize phase, the system waits a given time for the flow to reach slower speed required for analysis.. In the Run phase, cells are analysed and classified into histograms on the display in realtime. In the Count phase, cells are counted for a given volume. To finish the acquisition, click to the end icon or end button, or simply remove the sample tube from the sample port. The BioSafety cycle is started*. Status changes to Clean and then Ready. or To repeat an acquisition without removing the sample tube, click onto start icon or start button. To save results, click on the disk icon or select File- Save As. To analyse the next sample tube repeat steps on this page. * In case your FCM is not equipped with the BioSafety system, please inquire information for an add-on. 5

6 Operating Basics - Open the Instrument Settings Box Open the instrument settings box Make sure your previous measurement has been saved. or The instrument settings dialog box is used to optimize the acquisition for the particles of interest. The adjustments cover the gains of the optical detectors, e.g. the photomultiplier high voltages, the amplification mode (lin, 3 or 4 decade logarithmic), lower and upper level thresholds, and sample speeds. Instrument settings can be once set up for a given application and then be saved and reload for later use. Click the settings icon or select Acquisition - Instrument Settings... in the menu. The Instrument Settings Box appears. The instrument settings box 6

7 Operating Basics - Loading and Saving Instrument Settings To facilitate the selection of suited instrument settings, e.g. in case the system is used for multiple applications, instrument settings once saved can be reload. Loading of Instrument Settings In the Instrument Settings Dialog Box, click on "Load..." - The Open Dialog Box appears. Chose the instrument settings suited for your analysis. Click OK - The new instrument settings are loaded. Saving the Instrument Settings In the Instrument Settings Dialog Box, click on "Save..." - The Save Dialog Box appears. Enter a filename for the instrument settings. Click OK - The actual instrument settings are saved. 7

8 Operating Basics - Selecting Parameters for Analysis Before starting an acquisition, assure the parameters of interest are selected and set up according to your analysis. Opening the Parameter Setup Click the "Parameters..." button in the Instrument Settings box. The Parameter Setup Dialog Box appears. The Parameter Setup Dialog Box Parameter names cannot be changed after an acquisition. Only parameter labels can be changed. Only select parameters for acquisition that are required in order to 1. keep the data files as small as possible and 2. keep the acquisition as fast as possible. Parameter names Pulse Property Selections Lamp Excitation (Low Pass) 2 Wavelengths Signal Delay Check the parameter names are according to your optical setup. Typical names are shown in the example above. The parameter names are displayed on the histogram axis together with a parameter label. To change parameter names, click into the fields and enter the names by the keyboard. For each optical channel, you may analyse three pulse properties: 1. The pulse height, 2. the pulse area, and 3. the pulse width. Typically, only the pulse height is used for analysis. In case of a DNA analysis, the pulse area of the DNA parameter may be selected in order to discriminate cell doublets (say a doublet of G1 cells) from single cells (say one G2/M cell), which show up in the same DNA peak. For channels generating lamp excited signals, check this box. This may slighly improve the signal to noise ratio (and CVs) of the slower lamp signals. For channels used for analysis of signals generated by secondary light (typically the UV-lamp or a second laser) spot which is spatially separated from the primary light spot, check this box to compensate the signal delay due to the spatial separation. Select an appropriate delay (typically 50µs). 8

9 Operating Basics - Trigger, Labels, and Others Controls Parameter and Trigger enables Setup Select which parameter is used to trigger an event. In the left example, the check marks for parameters 1, 2, 4, 5, and 6 indicate that these parameters are enabled for the acquisition (by a selection in the parameter setup box). Parameter 3 will not be acquired. To select which parameter is to be used as the leading trigger parameter, click on the corresponding button until you see an asterix (*) on a green background. When no asterix is displayed, all parameters may trigger an event ("Trigger All"- mode). Parameter labels Parameter labels are used to show which cell property is currently associated with the parameter. Typical parameter labels are shown in the example on the left. Labels can be changed after an acquisition. Up / Down Control slow / fast slow / fast down / up To change the values of gain, sample speed, lower level (L-L) and upper level (U-L) thresholds, you can: Use the up/down control - click into the value field - to increase a value, click on the fast/slow up buttons - to decrease a value, click on the fast/slow down buttons Use the mouse - click into the value field - click onto an area without controls and hold the left mouse button down - move mouse to right/left to in-/decrease the value Directly enter the values by keyboard - click into the value field - enter the new value - click into another field 9

10 Operating Basics - Gain Controls Gain To change data scale or lin/log mode after the acquisition, see Calculate Parameters in the analysis part of the manual. Be sure not to use too high gain values. Increasing the gain values too much can cause a decrease of amplification or a loss of signals due to saturation effects. The gain value does not neccessarily be equal to the PMT high voltage nor does it be proportional to the amplification. Log Typical effect of change DNA distribution of bull sperms with X- and Y-chromosomes. DAPI and HBO lamp. Gain = 445. DNA distribution of bull sperms with X- and Y-chromosomes. DAPI and HBO lamp. Gain = EAT cells, linear amplification EAT cells, log 3 (3 decades) amplification Adjustment By means of the gain, the signal amplification can be adjusted in a wide range for each parameter separately. In case of photomultiplier tubes (PMTs), by changing the gain value, the PMT's high voltage (usually, between 0 and 1000 V) is adjusted. The gain value of a parameter can be increased (decreased) by clicking into the corresponding gain value field and using the Up (Down) buttons (see above). In case linear amplification is used, peaks are spreaded to the right (compressed to the left) when increasing (decreasing) the gain (see example on the left). In case logarithmic amplification is used, peaks are moved to the right (left) when increasing (decreasing) the gain. Use the gain to move the peaks of interest to a suited position in the histograms. Start with low gain values around When increasing the gain, make sure the most intense signals still appear in the histogram and are not moved out of scale. The gain value does not neccessarily be equal to the PMT high voltage nor does it be proportional to the amplification. Increasing (decreasing) the gain by 50 approximately causes an increase (decrease) of amplification by a factor of two. Gain values between 250 and 600 are a good start for adjustment. For each parameter, you can choose between linear or logarithmic amplification. In logarithmic amplification mode, the signal range can be set to 3 (log3) or 4 decades (log4), corresponding to a range of or Generally, a logarithmic amplification is advisable if particles with a broad range of intensities above 1:10 are to be analysed. Click the little arrow, select one of the following lin log3 log4 and click it. (linear) (logarithmic, 3 decade range) (logarithmic, 4 decade range) 10

11 Operating Basics - Sample Speed Speed For a high accuracy measurement, a low speed of 2.0 µl/s is advisable. Typical effect of change EAT cells, Speed = 3µl/s EAT cells, Speed = 20µl/s Adjustment By means of the speed value, the sample speed (in terms of µl/s) is set. The speed value of a parameter can be increased (decreased) by clicking into the corresponding gain value field and using the Up (Down) buttons (see above). The histogram is built up faster (slower). If the speed is increased too much, peaks in the histograms may become wider as a result of decreasing accuracy. If the speed is too low, particle sedimentation effects can influence a counting result. Typical Speed Values a) High accuracy measuremens (e.g. DNA with < 1% CV): Speed = 2.0 µl/s. b) Fast measurements and absolute counting: Speed = 5.0 µl/s 11

12 Operating Basics - L-L and U-L Thresholds Lower Level (L-L) Typical effect of change L-L = 10 L-L = 40 Adjustment Setting a proper lower level (L-L) avoids the acquisition of small and unwanted "noise" signals below a threshold. This allows the system have more time for the particles of interest. The L-L value of a parameter can be increased (decreased) by clicking into the corresponding L-L value field and using the Up (Down) buttons (see above). Start with a low L-L (e.g.10). Increase the L-L until no more of the small "noise" signals appear in the histograms. Make sure not to remove signals from particles of interest by a too high L-L. Upper Level (U-L) L-L and U-L cannot be adjusted after the acquisition. You may use 1P ranges or 2P polygons to gate out undesired signals instead. Setting an upper level (U-L) rejects signals above a threshold. The U-L value of a parameter can be increased (decreased) by clicking into the corresponding U-L value field and using the Up (Down) buttons (see above). Start with a high U-L (e.g. 999). Decrease the U-L until the unwanted intense signals disappear from the histograms. 12

13 Acquisition Setup - Preset Number of Events and/or Analysis Time You may preset the number of events to be acquired. Open the Acquisition Setup dialog: Click "Setup..." in the Instrument Settings Dialog - the Acquisition Setup Dialog is opened. In the Preset Count Gate drop down list, select a gate of interest or <none>, if the preset count is meant for the total number of events. Click in the Preset Count field and enter a new number of events value when the acquisition should stop. Make sure Enable for the Preset Count is checked. and/or Click in the Preset Time field and enter a new time value (in seconds [s]) when the acquisition should stop. Make sure Enable for the Preset Time is checked. Click OK - the preset values are selected. When both Preset Total Count and Preset Time are enabled, the acquisition will stop on the first occurance of any of the conditions, whichever comes first. 13

14 Acquisition Setup - Prerun, Stabilizing, and Clean Time You may optimize the time required for prerun, stabilizing and clean phase. The default values are appropriate for most applications. However, if you only have a very limited amount of sample or in case sample crosscontamination must be extremely low, you may need to use other time settings. Open the Acquisition Setup dialog: Click Setup... in the Instrument Settings Dialog - the Acquisition Setup Dialog is opened. Click in the Prerun Time, Stabilize Time or Clean Time field to enter a new time (in seconds [s]). Prerun Time Increasing the prerun time will assure the sample to appear at the flow cell at the start of an acquisition while increasing the sample uptake. Typical prerun times are 1-2 seconds. Stabilizing Time The stabilizing time allows the sample to reach it's final flow speed before the data acquisition starts. The time required for flow stabilizing depends on the final sample speed. Low final speed requires a longer stabilizing time. Typical stabilizing times are 2-10 seconds. Clean Time By increasing the clean time, the crosscontamination between different samples can be reduced. Typical clean times are 2-10 seconds. For demanding applications like leukocyte counting with very low concentrations (e.g. < 10/ml) or in case the particles have a high tendency to stick in the tubing, the clean time should be increased. 14

15 Acquisition Setup - Time Parameter Enable Time Parameter Enable the time parameter before starting the acquisition. The time parameter can be displayed in a 2 parameter dotplot similar to any other parameter. See "Selecting histogram type parameters for display". By means of the time-button, the acquisition of the time parameter is enabled. The time parameter allows to record a temporal course, e.g. in a kinetic analysis. The time parameter records the arrival time of each particle in the flow cuvette, in addition to the optical signals. The time parameter can be selected as one of the axes for a 2 parameter dotplot. The time axis always is linear during the aquisition. To switch on (off) the time parameter: 1. Click on the [+] button to enlarge the instrument settings box. 2. Click on the time button in the instrument settings box and make sure it is pressed (released). Adjust Time Range To select a time range appropriate to the kinetic experiment, open the Acquisition Setup dialog: Click "Setup..." in the Instrument Settings Dialog - the Acquisition Setup Dialog is opened. Click in the Time Range [s] field and enter a new value for the duration of the measurement or Click in the Time Resolution [ms] box and enter a new value for the time resolution. Click OK - the new time values are selected. Time Range [s] and Time Resolution [ms] depend on each other and cannot be set separately. Alternatively to the true time parameter, a event number parameter can be used even after an acquisition as a pseudo time parameter. See Calculate Parameters in the analysis part of the FloMax manual. 15

16 Sorting Sorting naturally is more complex than pure sample analysis. It requires good experimental skills and should be only performed by experienced and well trained operators. Before starting a sorting experiment, you should be well familiar how to perform a usual sample analysis. Select any gate for sorting. Enable / disable sorting by Sort button. By carefully selecting a suited particle concentration and sorting rate, the yield and purity of the sorted cells can be optimized. Adjustment Sorting of cells or other particles can be performed with the sorting flow cuvette (optional). By selecting one of the gates as the sort gate, the particles to be sorted can be choosen in the histograms by polygons, histogram ranges, quadrants or logical combinations thereof. To start an acquisition with sorting: 1. Adjust the acquisition settings and acquire some data for the sample. 2. Define a gate for the particles to be sorted in the resulting histograms. Refer to FloMax - Analysis how to define gates. 3. Select the gate for sorting. Click little arrow in the Sort Gate field and select a gate in the drop down box. 4. Enable (disable) sorting by clicking on the Sort button. 16

17 Controlling the Measurement - Start, Pause, Clear, End Button in acquisition dialog box Button in toolbar Starting a measurement A new measurement is automatically started when inserting a new tube to the sample port. If a tube is already inserted to the sample port, a click on one of the start buttons starts a new measurement. Pausing a measurement With the pause button, a measurement can be paused temporarily. To pause a measurement, click the pause button - the sample pump is stopped and the acquisition paused. To continue the measurement, click on start button. When removing a tube from the sample port, the BioSafety system is activated to prevent contamination by hazardeous material*. Simultaneously, a cleaning cycle is performed to avoid cross-contamination between individual samples. With the end buttons a running measurement is finished. To finish an acquisition click end button or remove sample tube from the sample port. Clearing histograms After changing acquisition settings, e.g. gain values, or after stabilizing of the flow, the histograms should be cleared. Click Clear button - the histograms are cleared. * In case your FCM is not equipped with the BioSafety system, please inquire information for an add-on. 17

18 Controlling the Measurement - Clean, Status Indication Button / control in acquisition dialog box Button in toolbar Cleaning flow cuvette and connection tubes By clicking the clean button, left-overs of the sample are flushed out of the flow cuvette and the connection tubes. Cleaning is automatically performed by the BioSafety system*. Additional clean cycles should be started manually at the end of the measurements, when cross contamination is critical. At the end of the day, the cleaning proceedure should be repeated 3-4 times. Click Clean button a clean message appears and a clean cycle is performed*. A new measurement is automatically started when inserting a new tube to the sample port. Status indication Do not insert a new sample tube onto the sample port during a clean phase. This could cause the new sample to be sucked out of the tube by the BioSafety system prior to analysis. Status Indication In the acquisition phase, the status indication marks one of the following situations: Prerun Stabilize Run Count Reposition Ready Clean sample tube is put under pressure system waits for flow to stabilize acquisition takes place absolute counting takes place sample pump is repositioned system is ready for acquisition a cleaning cycle takes place In conjunction with a sample loader automat, additional status indications are displayed. 18

19 Controlling the Measurement - Trigger Selection Control in acquisition dialog box If the trigger parameter does not generate valid signals (e.g. gain too low), no signals will appear in the histograms. Leading Trigger parameter By selection of a "leading - trigger parameter, the acquisition can be limited to those particles, which deliver a sufficient signal on that parameter. Typically, the forward scatter parameter (FSC) is selected as the trigger parameter. Depending on the application, selection of other parameters (SSC or fluorescence FL) for triggering can be more suited. For example for DNA measurements, triggering on the DNA parameter is advisable, since this parameter distinguishes between DNA containing and other particles. For microorganism detection, a sufficient intense fluorescence signal can be more clear to define a cell than a poor scatter signal. Selecting the trigger parameter Selecting parameter 1 as trigger parameter. Parameter 4 and 5 are disabled. Click the Enable button of the parameter you want to use to trigger, until a star on green background appears on the button. Make sure all other parameters which are to be acquired are enabled and show a check mark on their button. (See page 8 how to select parameters for acquisition.) Trigger on all parameters Instead of selecting one parameter for leading trigger generation, all parameters can allowed to generate a valid signal, independant of each other. This mode is called "trigger all". To select this mode: Click the Enable button of all parameters to be acquired until a check mark appears on all buttons. Selection of "trigger all" mode. (Parameter 4 and 5 are disabled.) When using fluorescence beads for coarse system adjustment, it can be a good help to select "trigger all" or a fluorescence parameter (e.g. FL1) as trigger. Using the FSC as trigger will cause all sufficiently big particles to be analysed. Dirt particles in the solution will then be analysed as well. This can be especially critical a) if the concentration of particles of interest is very low and b) if sheath fluid and dilution solutions are not filtered well enough. 19

20 Controlling the Measurement - Particle Rate, Count, Test Pulses Button / control in acquisition dialog box In case the particle rate is unexpectedly high, make sure gain, L-L and speed are adjusted properly. Make sure not too much "noise" is acquired. Display of particle rate This field displays how many cells or particles are analysed per second. Typical particle rates are below 300 1/s for high accuracy measurements and up to several thousands per second. The analysis rate depends on the flow speed and the particle concentration. Display of cell count The Count field displays how many cells are already analysed during the measurement. Optical test pulses As a simple system check, optical test pulses can be generated by a light emitting diode (LED) located in the optical bench. The test pulses will appear in histograms of the parameters which receive the light pulses. The pulses consist of four different pulse heights generated at a rate of about 1000 per s. System check with test pulses 1. Check the filter settings and make sure the detectors to be checked receive the light of the LED. 2. Select the parameters into a histogram. 3. Trigger on the parameter under test. 4. Click on [+] button to enlarge the instrument settings box. 5. Click on Test button to enable / disable the optical test pulse generation. Changing the size of the Acquisition Settings dialog box Click on + button to enlarge the Acquisition Settings dialog box. Click on - button to reduce the size the Acquisition Settings dialog box. Click on <- button to temporarily put the Acquisition Settings dialog box aside. Individually change the position and size by dragging the dialog box borders by the mouse. Move the mouse arrow onto a box border - double arrows are displayed. Hold mouse key pressed while dragging. Release the mouse key when finished. Closing the Acquisition Settings dialog box After finishing the acquisition or acquisition settings, click on the X button - the Acquisition Settings dialog box is closed. 20

21 Absolute Cell Counting - Analysing the Particle Concentration s When inserting a tube, make sure all three electrodes in the tube holder are covered by the sample. Make sure the sample still covers the upper electrode when the stabilize phase takes place. Make sure the sample does not contain bubbles which affect the volume sensing accuracy. Do not interrupt the counting phase by removing the tube or performing data analysis. Performing absolute counting For analysis of particle concentrations, i.e. counting the number of particles per ml, fill up sample tube with about 0.8 to 1 ml of the prepared sample suspension. Insert the sample tube onto the sample port. You should recognize a little "click" - the measurement starts and the status indication displays Prerun - Stabilize and then Run. When the sample releases the upper electrode, the volumetric absolute counting starts - the status indication displays Count. When the sample releases the lower electrode, the measurement is automatically finished - the status indication displays Ready. Standard counting volume is 0.2 ml. Other counting volumes, e.g. for rare events, are also available. Please contact your supplier. The concentration, i.e. the number of particles per ml, is shown in the result line on top of the histogram page. The result line also displays to total number of analysed particles. In case quadrants are used for the analysis, the concentration of particle-subpopulations is directly displayed in the quadrants. The concentration of particle subpopulations is displayed in the region statistics field. The Partec True Volumetric Absolute Counting system even allows to analyse the concentration of cell subpopulations selected after acquisition, provided a count phase has been run. Refer to the Analysis part of the FloMax Operating Manual for details. 21

22 Saving the Measurement File Menu Function Saving function Save button The save function will save the results in a file with the name given in the title bar: Click on Save button in the toolbar - the file is saved. or Click File in the menu bar - the file menu appears. Click Save in the file menu - the file is saved. A previously opened file is overwritten by the Save function. Generally, it is more save to use Save As function which always asks for a file name. In case no file name was specified yet, the Save As dialog box appears (see below). You may use long file names. Save As function The Save As function will ask for a file name before saving: Click File in the menu bar - the file menu appears. Click Save As... in the file menu - the Save As dialog box appears. In the Save in field, select the folder where the file is to be saved. In the File name field, enter a file name. Do not enter the extension - the file automatically will get the extension.fcs used for flow cytometry standard files. Click Save button - the file is saved. Save As dialog box Make use of different folders. E.g. use a different folder for each month or for each series of samples. This will help you when later locating your data. Button in toolbar You can also use the (optional) barcode reader to specify sample codes as file names. 22

23 Panels - Overview View templates, instrument settings, gating files and report templates have to be defined before they can be selected in panels. These settings are stored in files that may not be removed. Overview The panel system allows to define complete sets of measurements and run them in an automated way. This approach is often used in immunology to reduce the amount of user interaction. Usually, one complete panel consisting of svereral tubes is analysed for one patient sample. Using panels requires the definition of a panel once. Later this panel can be recalled for a panel acquisition or modified. Example panels are provided with the FloMax software. The main purpose of using panels is a) to automatically apply labels to the histogram axis, such as "CD3-FITC", b) to automatically save a series of measurements with a common basic name, such as "Smith001" for the first tube, "Smith002" for the next etc. Additionally FloMax panels have the feature to optionally allow automated c) printout of tube result after each measurement, d) selection of histogram templates for each tube, e) selection of special instrument settings for each tube, f) selection of special gating strategy for each tube, g) report generation for the complete panel (Word document). Tube 1 negative control IgG1-FITC / IgG2a-RPE Tube 2 T-helper cells CD3-FITC / CD4-RPE Tube 3 cytotoxic T-cells CD3-FITC / CD8-RPE Tube 4 NK cells CD3-FITC / CD16-PE Tube 5 T/B cells CD3-FITC / CD19-RPE Tube 6 cleaning tube Example: 2 Color / 5 Tube Panel for Standard Immunophenotype Analysis. 23

24 Panels - Definition Define / Edit a Panel 1. Select Panel - Edit Tube Panel... in the menu - the Panel Definition box is displayed. 2. Press Clear Panel to define a new panel. 3. Make sure the parameter names (FSC, SSC, FL1 etc.) are correct. If not, first enter correct names as decribed on page. Panel Definition box Usually, view template and instrument settings stay the same for all tubes of a panel. Therefore, only select these for the first tubes. Clear these settings for the other tubes - they will then use the settings used for the first tube. Gating settings can be included in view templates as well. This is the prefered way since it also stores how gating is applied to the histograms. To remove a tube from the panel, select the tube in the tube list and press Remove Tube. Use a separate folder to store the panels, e.g. C:\FloMax\Panels. Define and Add Tube 1. Enter a short description for first tube, e.g. "neg. control". 2. Enter the tube position (only required if the panel is going to be used with the sample automate). 3. Enter axis labels for the tube, e.g. IgG1-FITC for FL1 and IgG2a-RPE for FL2. These labels will be displayed on the histogram axis together with the parameter names. 4. Check Save for an automated file saving at the end of the acquisition. 5. Check Print for a automated printout of the tube result after acquisition. 6. Select a view template for an automated selection of a histogram layout/page (including regions and gates). Clear the view template to use the actual histogram layout. 7. Select instrument settings for an automated selection of special instrument settings for the tube. Clear instrument settings to use the actual instrument settings. 8. Select a gating file for an automated load of gates and regions for the tube. Clear gating to use the actual gating settings (or the gates setup in the view template). 9. Press Add Tube - the tube (with description) now is defined and appears in the tube list. 10. Repeat steps starting from 1. for all other tubes of the panel. Automated Panel Report For an automated generation of a complete panel report, select a report form and check Create Report. Save Panel Press Save... - a Save As dialog appears. Specify a folder and filename for the panel. As extension, ".pnl" is added automatically for panel files. Load Panel Press Save... - a Save As dialog appears. Specify a folder and filename for the panel. As extension, ".pnl" is added automatically for panel files. 24

25 Panels - Acquisition Prepare the Acquisition 1. Select Acquisition - Run Tube Panel... in the menu - a Save As box is displayed. 2. Specify a folder and basic file name for the panel, e.g. "Smith" (or use a barcode reader to enter a sample code). 3. Press Open to start with the panel - the Panel Definition box is displayed. 4. Check the panel or load another predefined panel. Make corrections as neccessary. 5. Press OK - the Instrument Settings box is displayed. Histogram template, instrument settings, gating strategy are set if specified in the panel. If not specified, the actual settings are used. Run the Panel Acquisition 1. Insert the first sample tube on the sample port - the tube is analysed. The histogram axis displays the labels specified for that tube. The filename is automatically set. Specify basic file name and folder for the panel 2. Tube Acquisition is finished when a) the specified number of events is reached, b) a specified time is exceeded, c) removing the sample, d) the sample runs out, e) selecting acquisition end manually - if Save was checked for that tube, the file is saved as "Smithnnn" in the spcified folder. The tube number is nnn, e.g "Smith001" is the filename for the first tube. Panel Definition box 3. Press Next in the Tube box of the instrument settings. 4. Repeat from 1. for the other tubes. Tube control in instrument settings box Select Prev. to repeat acquisition of a previous tube. Panel Report If a suited Word report template is specified and checked for the panel, when finishing the last tube of the panel, a multi-tube multi-page report is generated. This may require some seconds to complete. The report can then be edited and completed in Word. 25

26 Panels - Optimizing Optimizing Panels - Step by Step I. Optimize the report template 1. Load a basic view template, e.g. "Report.dot" (or another template based on this). 2. Change the report template according to your requirements. 3. Save the view template using a unique name, e.g. "2Color5TubeReport.dot". Report templates must be based on another report template, e.g. "Report.dot" in order to contain the required report macro. Use unique folders for reports, panels, view templates, instrument settings and so on, e.g. "C:\FloMax\Panels". II. Optimize the view template 1. Make sure the view template fits to the report template. 2. Save these view template using a unique name, e.g. "2ColorForm.FCS" Report templates assume histograms and region statistics to appear at a specific location. Therefore, view templates have to be designed carefully in order to fit to the report templates. III. Set up the instrument settings 1. Set up parameter enables, trigger, gain values, lower level -values, while measuring a (typical) sample. 2. Save these instrument settings using a unique name, e.g. "2ColorSetup.ins" IV. Prepare the panel 1. Enter the labels for each tube. 2. Select view template and instrument settings for the first tube only. 3. Make sure the "Save" check is on for each tube. 4. Save the panel file (e.g. "2ColourPanel.pnl"). V. Run the panel acquisition a) Prepare the panel acquisition 1. Select Acquisition - Run Panel Acquisition Enter the sample code (e.g. the patient name "Smith" or barcode by a barcode reader) 3. Select a folder where to save the panel files. 4. Check the panel is correct. Use unique folders for panel results for a series of tube measurements, e.g. "C:\FloMax\Results\Smith". b) acquire tube no. 1 (find lymphocytes) 1. Insert the tube no. 1 for acquisition. It's file will have the name "Smith001.FCS". 2. Correct the gain- and ll-values if neccessary - the file is automatically saved on end of the acquisition. 3. Select the lymphocyte region. 4. Save the file again by clicking on the save icon (save it together with the region). c) acquire tube no. 2 (negative control) 1. Press the "Next" tube button. 2. Insert tube no. 2. for acquisition 3. Correct the gain- and ll-values if neccessary - the file is automatically saved on end of the acquisition. 4. Select the quadrant region and apply the lymphocyte region as the FL gate. 5. Save the file again by clicking on the save icon (save it together with the gates). d) acquire other tubes (immune status tubes) 1. Press the "Next" tube button. 2. Insert next tube for acquisition - the file is automatically saved on end of the acquisition, together with the results. When the last panel tube is finished, the panel report is generated automatically. 26

27 Life Gating Applying gates or regions 1. Double click the histogram for which a gate is to be applyed - the histogram property box is displayed. 2. Click the little arrow in the gate drop down list and select the gate or region. 3. Press OK - the gate is applied and the histogram property box is closed. If a gate is applied to a histogram, this is displayed at the top of the histogram. Removing gates or regions from a histogram 1. Double click the histogram for which a gate is to be applyed - the histogram property box is displayed. 2. Click the little arrow in the gate drop down list and select the <None>. 3. Press OK - the gate is removed from the histogram and the histogram property box is closed. 2P dotplot of FL1 - FL2, gated by a polygon region "Lympho" defined in the FSC - SSC dotplot. Gating can be applied during or after an acquisition: online (or life) gating and offline gating. In any case, all events are saved. This allows to completely reanalyse the measurement, starting from the original data. 27

28 Crosstalk Compensation - Realtime Online Compensation Compensation values can be active also for parameters not displayed in histograms. Make sure compensation is only selected for required parameters. Tip: Press Clear All in the compensation box to set all compensation values to zero before setting up the compensation. Set up Realtime Compensation for Acquisition 1. Open the instrument settings box. 2. Acquire some uncompensated data for a suited negative-positive control sample. Make sure N- Color Compensation button in the instrument settings box is off (out). 3. Press Pause to temporarily stop the acquisition. 4. Select Analysis - Compensate Crosstalk... to open the compensation toolbox. 5. Load predifined compensation or set compensation values graphically or by keyboard Refer to FloMax Analysis for details. 6. Press Compensate in the compensation toolbox - data analysed up to now is compensated. 7. Press N-Color Compensation in the instrument settings box. 8. Press Start to restart the acquisition - data acquired from now on will be displayed compensated. 9. Close compensation box if no more needed. Activation of Realtime N-Color Compensation in the instrument settings box. 28

29 Open a Measurement File Menu Function Open dialog box Button in toolbar Open button Open function The open function will reload the results which have been saved in a file before. Make sure any previous file was saved. Click on Open button in the toolbar - the Open dialog box appears. or Click File in the menu bar - the file menu appears. Click Open... in the file menu - the Open dialog box appears. In the Look in field, select the folder where the file is located. Click on the file to be opened. The extension is.fcs. Click Open button - the file is opened and the results are displayed. In case you forgot in which folder the file was located, you may use the Windows search files function in the Start menu. Refer to FloMax Operating Manual Analysis about analysing files. 29