Tutorial: FCAP Array Software with BD FACSArray Bioanalyzer

Transcription

1 Tutorial: FCAP Array Software with BD FACSArray Bioanalyzer After completing this tutorial you will be able to: Create an Experiment with the Experiment Wizard in FCAP Array software. Export the Experiment and use it to acquire data with BD FACSArray bioanalyzer. Analyze data files within the FCAP Array software Rev. A FCAP Array Software Tutorial 1

2 Materials Check with your local sales representative for specific product information. Product part numbers and pricing information are also available in the online catalog at For additional information regarding BD CBA Flex Set Beads, see BD FACSArray bioanalyzer BD FACSArray system software FCAP Array software two 96-well plates (one for instrument setup, one for the bead assay) BD CBA Human Soluble Protein Master Buffer Kit NOTICE Consult your BD CBA Human Soluble Protein Master Buffer Kit Manual for additional materials required for preparing samples. BD CBA Flex Set Beads: Human IL-2 Human IL-4 Human IL-5 Human IL-6 Human IL-8 Human IL-1β Human IL-10 Human TNF Human IL-12p70 Human IFN-γ References BD FACSArray Bioanalyzer User s Guide BD FACSArray System Software User s Guide BD CBA Human Soluble Protein Master Buffer Kit Manual FCAP Array Software User s Guide 2 FCAP Array Software Tutorial Rev. A

3 BD FACSArray Training Recommended Training BD FACSArray interactive training CD-ROM (ships with instrument). Additional Training The BD FACSArray Workshop or on-site training is available for BD FACSArray bioanalyzer operators. For more information about the content, dates, and locations for the BD FACSArray Workshop or on-site training, call BD Biosciences Customer Training at Basic Terms The following terms are used (with capitalization as shown) throughout this tutorial to indicate specific constructs, procedures, or concepts. Term bead Bead bead assay cluster experiment Experiment plex Plex sample Description An analyte-specific capture particle with distinctive, discrete fluorescence characteristics (all instances of a bead bind the same analyte and have the same fluorescence characteristics). An FCAP Array software construct that specifies the analyte binding and fluorescence characteristics of a bead. A method of using beads and reporter antibodies to detect or measure the concentration of analytes in a sample. A population of events in a bead assay data file. Each cluster corresponds to a bead, therefore, the events in a cluster have a distinctive, discrete fluorescence characteristic. A bead assay. A BD FACSArray system software or FCAP Array software construct that defines the Plex(es), standards, controls, and test samples for a bead assay. One or more beads used in a bead assay. An FCAP Array software construct that specifies a plex, a standard set, and a Bead-cluster assignment. One of the source solutions to which capture beads and reporter antibodies are added in a bead assay. A sample can be a test, a standard, or a control Rev. A FCAP Array Software Tutorial 3

4 Bead Assay Overview Bead assays determine concentrations for multiple analytes (example: proteins and peptides) in a sample. In a bead assay, one or more beads with discrete and distinct fluorescence intensities are used to simultaneously detect multiple analytes in a small sample volume. The beads capture and quantify soluble analytes through a sandwich schema. capture antibody analyte reporter antibody fluorescent molecule bead A particular analyte in the sample binds to a corresponding bead with given fluorescent characteristics. The bead is coated with capture antibodies specific for that analyte. A reporter antibody (different from the capture antibody) binds to the analyte. The reporter antibody is conjugated with fluorescent molecules (different color from those used to distinguish beads). Unbound (excess) reporter antibodies and analytes are eliminated by washing. Samples are acquired with a flow cytometer and acquisition software capable of saving data in a Flow Cytometry Standard (FCS) 2.0 file format. FCAP Array software analyzes the FCS 2.0 files. Bead populations are found by clustering, and assigning beads to clusters. Reporter antibody florescence intensity within a population measures analyte-specific binding. Quantitative Analysis Quantitative analysis determines analyte concentrations based on known concentration values of a set of standards. With FCAP Array software, analyte concentration can be determined for many analytes (beads) per sample. The number of concentration levels used to calibrate the standard curve and the number of sample and standard replicates are easily adjusted. FCAP Array software reads the FCS 2.0 data files from an Experiment, locates clusters (to which analytes have been assigned), and then determines the median fluorescence intensity (MFI) of the detector antibody for each analyte. The software fits a standard curve to the data from the concentration standards. The curve used to fit the data is selected from several mathematical models. The standard curve is used to calculate concentration values for each of the measured analytes in each sample. 4 FCAP Array Software Tutorial Rev. A

5 Introduction The BD FACSArray bioanalyzer and software can be integrated with FCAP Array software to encompass the entire bead assay procedure, from instrument setup to analysis report. This tutorial follows the recommended setup, acquisition, and analysis workflow for performing BD CBA Flex Set bead assays with the BD FACSArray bioanalyzer. It consists of the main steps shown in the following figure (steps that are performed using BD FACSArray system software are displayed with a thicker border). Set Up Instrument Transfer Template Create FCAP Experiment Export Experiment Import Experiment Acquire Data Analyze Data These main steps are covered in the following tutorial sections: Performing Instrument Setup Perform instrument setup on the BD FACSArray bioanalyzer. Save the instrument setup Experiment as an Experiment Template in BD FACSArray system software. Transferring the Experiment Template Transfer the template file from the BD FACSArray templates folder to the FCAP Array Templates folder. Creating an Experiment in FCAP Array Software Create an FCAP Array software Experiment to be used with the BD FACSArray bioanalyzer. Skip the Plex cluster assignment. Exporting the Experiment File Export the Experiment file from FCAP Array software. Importing Experiment File into BD FACSArray Software Import the Experiment file to BD FACSArray system software. Acquiring Data Using the BD FACSArray bioanalyzer and software, acquire BD CBA Flex Set sample data and export the FCS 2.0 list-mode data files. Analyzing Data Using FCAP Array software, assign data files to Experiment samples. Analyze the data, review the results, and finalize the Experiment Rev. A FCAP Array Software Tutorial 5

6 Performing Instrument Setup In this section, you perform instrument setup on the BD FACSArray bioanalyzer to optimize instrument performance for BD CBA Flex Set bead assays: detector voltages are set acquisition gates are defined optical spillover corrections are determined You save the instrument setup Experiment as an Experiment template in BD FACSArray system software. The Experiment template is used with subsequent bead assay Experiments to ensure optimal voltages, gates, and spillover corrections are used. NOTICE It is recommended that the instrument setup procedure is performed monthly for each BD FACSArray bioanalyzer used with BD CBA Flex Set Experiments. In addition, the setup procedure should be run after any instrument maintenance, including syringe replacements. Set Up Instrument Transfer Template Create FCAP Experiment Export Experiment Import Experiment Acquire Data Analyze Data Performing Instrument Setup a Creating an Instrument Setup Experiment b Setting Instrument Voltages c Correcting for Optical Spillover d Saving the Instrument Setup Experiment as a Template Creating an Instrument Setup Experiment In this section, you use the BD FACSArray Experiment Wizard to create and configure an instrument setup Experiment. 1 Start up the BD FACSArray bioanalyzer (if needed) and launch BD FACSArray system software (if needed) as specified in the BD FACSArray System Software User s Guide. 2 Click on the Experiment Wizard tool ( ) in the application toolbar. The initial view of the Experiment Creation Wizard is displayed (Figure 1-1 on page 7). 6 FCAP Array Software Tutorial Rev. A

7 3 Select Default and click Next. Figure 1-1 Experiment Creation Wizard: Selecting a Wizard Session view The Experiment Creation Wizard advances to the next view. 4 From the drop-down menu, select BD CBA Flex Set and click Next. NOTICE In subsequent setups you can select the template you create in this section. The Experiment Creation Wizard advances to the next view Rev. A FCAP Array Software Tutorial 7

8 5 Select Yes (to reserve wells to check instrument settings and to correct for optical spillover). Enable NIR (APC-Cy7) and Red (APC, Alexa Flour 647) by marking their checkboxes. Click Next. 6 Select the default settings in the Enter the number of Calibrator Standard Replicates view, and in the five subsequent views, by clicking Next six times, until you reach the Adjusting the plate layout view. 7 Select Contiguous Horizontal (for a plate layout without separators) and click Next. The Experiment Creation Wizard advances to the next view. 8 FCAP Array Software Tutorial Rev. A

9 8 In the Loader Settings view, change the Sample Flow Rate to 1.0 µl/sec, and click Next. The Experiment Creation Wizard advances to the next view. 9 Accept the default values in the Acquisition Settings view by clicking Next to advance to the Experiment Name view. 10 Name the Experiment BD CBA Flex Set Setup mmddyy (in place of mmddyy, enter today s date), and click Next. The Experiment Creation Wizard advances to the next view Rev. A FCAP Array Software Tutorial 9

10 11 Click Next in the Saving Wizard Session (you do not need to save the wizard session). The Experiment Creation Wizard advances to the final view. 12 Review the wizard selections and click Finish. The specified Experiment is created and it opens in the Prepare workspace. 10 FCAP Array Software Tutorial Rev. A

11 Setting Instrument Voltages Preparing Setup Bead Samples In this section, you prepare the instrument setup bead samples in a microwell plate. NOTICE The Experiment Wizard has created a plate template with 12 wells (the minimum number of wells it can define). In the instrument setup Experiment, you use only 5 wells of the plate: A1 through A5. 1 Add 175 µl of Wash Buffer to each of the wells A1, A2, A3, A4, and A5 of a microwell plate. 2 Add 25 µl of Instrument Setup Bead F1 to well A1. 3 Add 25 µl of Instrument Setup Bead A1 to well A2. 4 Add 25 µl of Instrument Setup Bead F9 to well A3. 5 Add 25 µl of Instrument Setup Bead A9 to well A4. 6 Add 25 µl of PE Instrument Setup Bead F1 to well A5. 7 Shake the plate using a plate shaker at 500 RPM for 20 seconds Rev. A FCAP Array Software Tutorial 11

12 Adjusting Loader Settings 1 If needed, click the Prepare tool ( ) in the application toolbar to display the Prepare workspace. 2 In the Plate editor, click well A4 (see figure). 3 With the Inspector window, select the Acq tab and adjust the loader settings as follows (see figure). Sample Flow Rate: 0.5 µl/sec Sample Volume: 100 µl Mixing Volume: 60 µl Mixing Speed: 200 µl/sec Number of Mixes: 1 Wash Volume 200 µl NOTICE The Sample Flow Rate is changed to 0.5 µl/sec to provide the additional time needed to complete adjustments using well A4. 12 FCAP Array Software Tutorial Rev. A

13 4 In the Plate editor, click well A5. 5 With the Inspector window, select the Acq tab and adjust the loader settings as follows (see figure). Sample Flow Rate: 0.5 µl/sec Sample Volume: 60 µl Mixing Volume: 60 µl Mixing Speed: 200 µl/sec Number of Mixes: 1 Wash Volume 200 µl NOTICE The Sample Flow Rate is changed to 0.5 µl/sec to provide the additional time needed to complete adjustments using well A Rev. A FCAP Array Software Tutorial 13

14 Adjusting Scatter and Far Red Voltages 1 Click the Setup tool ( ) in the application toolbar to display the Setup workspace. 2 Click on the Parameters tab in the Instrument window to display the voltage settings for each parameter. a b Adjust voltages as shown in the figure. Verify that Log is enabled (checkbox is marked) for each parameter. 14 FCAP Array Software Tutorial Rev. A

15 Adjusting Voltages with Well A4 In this section you load the plate and acquire from well A4 to adjust the voltages for NIR-A and Red-A. 1 Place the microwell plate containing the setup beads in the loading tray of the BD FACSArray bioanalyzer so that well A1 is aligned over the A1 position label on the plate holder. 2 With the Plate editor, click Load in the Plate control group. The Status box should display the following message Rev. A FCAP Array Software Tutorial 15

16 3 In the Select control group, click None to deselect the wells, then click well A4 in the Plate view. Tip To complete the setup without adding more sample to well A4, steps 4 through 11 below must be performed in a limited time (about 3 minutes). Study these steps in advance to minimize the time needed to consult instructions during the procedure. Tip To improve the refresh rate of the fluorescence statistics, reduce Events to Display to 50 evt prior to starting step 4. 4 Click Setup in the Acquisition control group. The A4 well is highlighted. Events appear in the Template plots, but data is not saved. 16 FCAP Array Software Tutorial Rev. A

17 5 In the Template window, move the P1 gate over the singlet population in the FSC-A vs SSC-A dot plot Rev. A FCAP Array Software Tutorial 17

18 6 Right-click in the statistics box (see figure) and select Edit Statistics View. The Edit Statistics View dialog is displayed. 7 Select the Populations tab. Mark the checkboxes as shown in the figure so that only P1 #Events and P1 %Parent are enabled. 18 FCAP Array Software Tutorial Rev. A

19 8 Select the Statistics tab. Mark the checkboxes as shown in the figure so that only the Yellow-A, NIR-A and Red-A Median and the Yellow-A Mean statistics are enabled. 9 Verify that Sort by Parameter is selected. 10 Click OK. The Edit Statistics View dialog is dismissed and the Statistics box displays the selected statistics Rev. A FCAP Array Software Tutorial 19

20 11 Adjust the voltages for A4: a b c d Click the Parameters tab of the Instrument window (if needed). Adjust the NIR-A voltage setting until the Median fluorescence for population P1 in the NIR-A channel is in the range 160,000 ±2000. Adjust the Red-A voltage settings until the Median fluorescence for population P1 in the Red-A channel is in the range 160,000 ±2000. Click Setup in the Acquisition control group to stop the acquisition. Figure 1-2 Adjusting NIR-A and Red-A voltage settings NOTICE i ii iii iv v If you run out of sample before completing the adjustments: Click Unload in the Plate control group. Add 175 µl of wash buffer and 25 µl of Instrument Setup Bead A9 to well A4. Ensure that the new bead sample is well mixed. Click Load in the Plate control group. Click None in the Select control group to deselect the wells, then click well A4 in the Plate view. Click Setup in the Acquisition control group to start setup acquisition of well A4. 20 FCAP Array Software Tutorial Rev. A

21 Adjusting Voltages with Well A5 In this section you acquire from well A5 to adjust the voltages for the Yellow-A detector. 1 With the plate editor: a b In the Select control group, click None to deselect the wells. Click well A5 in the Plate view. c Click Setup in the Acquisition control group to start acquisition of well A5. 2 As the sample is being acquired, adjust the voltages for Yellow-A: a b c Click on the histogram plot parameter label and use the drop-down menu to change the histogram parameter to Yellow-A (see figure). Adjust the voltage setting for Yellow-A until the Mean fluorescence for population P1 in the Yellow-A channel is in the range 60 ±5. Click Setup in the Acquisition control group to stop acquisition Rev. A FCAP Array Software Tutorial 21

22 Correcting for Optical Spillover In this section, you acquire from wells A1, A2, and A3 to provide data for calculating optical spillover corrections. After the spillover calculation, you acquire from well A5 to complete the setup Experiment. 1 Click Acquire in the Acquisition control group to begin acquisition of the optical spillover wells. 2 If a gating error dialog is displayed, click OK. 3 Click the Analyze tool ( ) in the application toolbar to display the Analyze workspace. 4 With the Plate editor, click well A1. 22 FCAP Array Software Tutorial Rev. A

23 5 In the Template window, adjust the singlet and histogram gates as necessary to encompass populations as shown in the figure. 6 Click well A2. Adjust singlet and histogram gates as necessary to encompass populations as shown in the figure Rev. A FCAP Array Software Tutorial 23

24 7 Click well A3. Adjust singlet and histogram gates as necessary to encompass populations as shown in the figure. 8 Select Instrument > Calculate Spillover. When the spillover calculation has completed, the Spillover dialog is displayed. 9 Click OK. 24 FCAP Array Software Tutorial Rev. A

25 10 Click the Setup tool ( ) in the application toolbar to display the Setup workspace. 11 With the Plate editor: a b In the Select control group, click None to deselect the wells. Click well A5 in the Plate view. c d Click Acquire in the Acquisition control group. When acquisition completes, click Unload in the Plate control group, and unload the setup plate. Saving the Instrument Setup Experiment as a Template In this section, you create and save the BD FACSArray setup Experiment template. To complete the setup Experiment configuration, you must close and reopen the Experiment. NOTICE If you save an Experiment template without first closing and reopening the Experiment, it is possible that the stopping gate criteria will not be properly recorded in the template file. 1 Click the Prepare tool ( ) in the application toolbar to display the Prepare workspace. 2 Double-click the icon of the currently open setup Experiment in the Browser pane. o The Experiment closes Rev. A FCAP Array Software Tutorial 25

26 3 Double-click the icon of the now closed setup Experiment in the Browser pane. o The Experiment reopens. 4 Select Experiment > Save As Template from the menu bar. The Save Experiment as Template dialog is displayed. 5 Name the template BD CBA Flex Set Setup mmddyy (in place of mmddyy, enter today s date), and click OK. Instrument setup is now complete. If desired, you can exit the BD FACSArray system software. 26 FCAP Array Software Tutorial Rev. A

27 Transferring the Experiment Template After a modified BD CBA Flex Set Experiment template has been saved, it should be made available to FCAP Array software by copying the template to the FCAP Array software Templates folder. FCAP Array software uses the template when it exports an Experiment back to BD FACSArray system software (see Exporting the Experiment File on page 44). Set Up Instrument Transfer Template Create FCAP Experiment Export Experiment Import Experiment Acquire Data Analyze Data 1 Copy the modified BD CBA Flex Set template XML file: a Using the folder system of your BD FACSArray workstation, open the folder: C:\ Program Files\ BD FACSArray System Software\ templates\ BD CBA Flex Set Setup mmddyy b Copy the file BD CBA Flex Set Setup mmddyy.xml by right-clicking the file icon and selecting Copy from the context-sensitive menu. Close the folder. 2 Paste the.xml template file to an appropriate location in the computer containing FCAP Array software: a Open the folder: C:\ Program Files\ Soft Flow\ FCAP Array v. 1.0\ Templates b Paste the template XML file by right-clicking in the file list and selecting Paste from the context-sensitive menu. Close the folder Rev. A FCAP Array Software Tutorial 27

28 Creating an Experiment in FCAP Array Software Before you can acquire sample data, you must create an FCAP Array software Experiment. The Experiment is exported and used to acquire data with the BD FACSArray bioanalyzer. Set Up Instrument Transfer Template Create FCAP Experiment Export Experiment Import Experiment Acquire Data Analyze Data Creating an Experiment in FCAP Array Software a Using the Experiment Wizard b Entering Test Samples and Dilutions c Creating a New Plex d Using the Bead Library e Choosing Plex Components f Specifying Standards g Creating a Plate Layout h Ending the Experiment Wizard Session 1 To launch FCAP Array software, do either of the following: Double-click the FCAP Array software shortcut icon on the desktop Select Start > Programs > FCAP Array > FCAP Array As the software launches, the Login to FCAP Array dialog is displayed (see figure). 2 Enter your user name and password and click Log in. NOTICE For this tutorial, you need both administrative and signing privileges. When FCAP Array software is installed, a user name Administrator is provided, with password welcome. Administrator has both administrative and signing privileges. 28 FCAP Array Software Tutorial Rev. A

29 If the login is successful, one of the following is displayed: the Quick Start dialog the General Information view of the main activity window Using the Experiment Wizard In this section, you use the Experiment Wizard to create an Experiment document. 1 Do one of the following to start an Experiment Wizard session. If the Quick Start dialog is displayed, click the New experiment icon. Otherwise, click the New Experiment icon in the application toolbar: The Experiment Wizard is displayed Rev. A FCAP Array Software Tutorial 29

30 Entering Test Samples and Dilutions In this tutorial, you acquire and analyze two test samples. In the Test Samples and the Dilutions and Replicates views, enter information about your test samples. 1 Click Next to advance to the Test Samples view. In the Test Samples view, you specify the number of test samples, and provide sample names. 2 Specify 2 for Number of samples. Tip If you type in the value, press the Enter key. Default sample names are displayed for 2 test samples. In the next step, you modify the test sample names. 3 Change the test sample names to Tutorial001 and Tutorial002. a b c d Click the first line to highlight it. Click again on the first test sample name to obtain an editable field. Edit the first test sample name and press Enter to advance to the next line and obtain an editable field. Edit the second test sample name and press Enter. 30 FCAP Array Software Tutorial Rev. A

31 4 Click Next to advance to the Dilution and Replicates view. NOTICE The default dilution factors is Change the dilution factor for test sample Tutorial002 to 2.0. a b Select Tutorial002 from the drop-down menu (see figure). Enter 2.0 for the Dilution factor Rev. A FCAP Array Software Tutorial 31

32 Creating a New Plex (For definitions of terms used in this section, see Basic Terms on page 3.) If you just installed FCAP Array software, your Plex and Bead libraries are empty. Before you can create a Plex, you must enter the Beads needed by the Plex in your Bead Library. 1 Click Next to advance to the Selecting Saved Plex view. 2 Verify that **New Plex** is selected in the browser window. 32 FCAP Array Software Tutorial Rev. A

33 3 Click Next to advance to the Plex Components view. In the Plex Components view, you specify the Beads for your Plex. If you just installed FCAP Array software, there are no Beads to choose from (see figure). To create the Beads needed for your Plex, use the Bead Library as described in the next section. Figure 1-3 Plex Components Using the Bead Library In this section, you enter new Beads in the Bead Library. With the Bead Library, you also create a group for the Beads in your plex. 1 Click Edit to display the Bead Library dialog (see Figure 1-4 on page 34). 2 Enter Human IL-2 for Analyte ID. 3 Enter A4 for Bead ID. 4 Click Add. The new Bead is added to the Bead library list on the left Rev. A FCAP Array Software Tutorial 33

34 5 Repeat steps 2 through 4 for each row in the table below (first row is done). Analyte Human IL-2 Human IL-4 Human IL-5 Human IL-6 Human IL-8 Human IL-1β Human IL-10 Human TNF Human IL-12p70 Human IFN-γ Bead ID (cluster location) A4 A5 A6 A7 A9 B4 B7 D9 E5 E7 Tip Catalog# and Lot# can also be specified for each Bead, but they are not required for running an assay. Figure 1-4 Bead Library Tip Another way to populate the bead library is to download bead definitions from the CBA Flex Set website at 34 FCAP Array Software Tutorial Rev. A

35 6 Click Edit Groups to display the Bead Groups dialog. If needed, click the + box to display the Analytes and Bead IDs for All Beads. Figure 1-5 Bead Groups dialog 7 Click New Group to display the New Bead Group dialog. 8 Enter Cytokine 10-Plex as the name of the new group and click OK. Figure 1-6 New Bead Group dialog The Bead Group dialog is displayed Rev. A FCAP Array Software Tutorial 35

36 9 Choose all the Beads to be included in the Cytokine 10-Plex group by marking their checkboxes (consult the table following step 5 on page 34 for a list of the Beads to be included). 10 Click OK to return to the Bead Groups dialog. The Cytokine 10-Plex group is now included in the browser listing of the Bead Groups dialog. 36 FCAP Array Software Tutorial Rev. A

37 11 If needed, click the + box to the left of the new group to open it. Verify that the correct 10 Beads are in the group. 12 Click OK to return to the Bead Library dialog. 13 Click OK to return to the Plex Components view of the Experiment Wizard. Choosing Plex Components Now that there are Beads in your Bead Library, you can specify the Beads needed by your Plex. Because you defined a group for the Plex Beads, a shortcut allows you to do this in one step. 1 Double-click the Cytokine 10-Plex Bead group folder in the Beads listing on the left side of the Plex Components view. All 10 Beads from the group are added to the Selected beads listing on the right (Figure 1-7 on page 38). Tip Beads can also be selected one at a time using the button Rev. A FCAP Array Software Tutorial 37

38 Figure 1-7 Plex Components view after selecting Beads NOTICE Because this FCAP Array software Experiment is being created prior to data acquisition (design first workflow), data is not available for completing the next view: Clustering Parameters. Therefore, skip both the Clustering Parameters view, and the associated Analyte Assignment view for now, and return to these activities after acquisition (see Assigning Beads to Clusters on page 59). 38 FCAP Array Software Tutorial Rev. A

39 Specifying Standards In this section, you specify the fitting equation and standards for a quantitative assay. 1 Click Next twice to advance to the Qualitative/Quantitative view. 2 Verify that Quantitative is selected and Uniform fitting equations is enabled. 3 Click in the table cell in the top row under Fitting equation to activate its pull-down and select 4 parameter logistic. The table displays 4 parameter logistic as the fitting equation for every Bead-cluster Rev. A FCAP Array Software Tutorial 39

40 4 Click Next to advance to the Standards view. In the Standards view for a Quantitative assay you specify how many standards are to be used, and the concentration and the number of replicates for each standard. 5 Specify 10 for Number of standard samples and verify that Uniform concentrations for all analytes is enabled. Keep replicate values at 1. 6 Click in the table cell of the Unit row and CC column to activate the units drop-down menu. Select pg/ml for the concentration unit. 7 Enter the standard sample concentrations as shown in the table below. Tip Press the Enter key after typing a value to advance to the next cell in the CC column. Standard Sample Concentration Standard Sample Concentration Std Std Std Std Std Std Std Std Std Std When you have completed entering the concentrations, verify that your Standards view looks exactly like the figure. Be sure to check that you specified a Unit in step FCAP Array Software Tutorial Rev. A

41 NOTICE In this tutorial you do not use Controls and Reporting Messages. Therefore, skip over the next two views, and proceed to the Plate Layout Options view. Creating a Plate Layout In this section, you specify your sample arrangement. The acquisition order specified in this section should match your microtiter plate layout. BD FACSArray system software acquires data according to the plate layout specified in FCAP Array software. 1 Click Next three times (once on each of three consecutive views) to advance to the Plate Layout Options view. 2 Verify that 96 well plate is selected. NOTICE The BD FACSArray bioanalyzer is not compatible with any other Plate/ Rack format. 3 Select Row by row as the well assignment method. 4 Enable the Place samples at the end option (by marking its checkbox). This option places the test samples after the standard samples in the plate layout. 5 Enable the Standards/samples start on a new row/column option. This option puts the test samples at the start of a new row. o Rev. A FCAP Array Software Tutorial 41

42 Ending the Experiment Wizard Session Next, you name the Experiment and complete the Experiment Wizard session. 1 Click Next to advance to the Experiment Name view. 2 Enter Tutorial as the Experiment name. o 3 Click Finish to close the Experiment view. The new Experiment opens in the main window in the Plate Layout view, and a Save As dialog is provided to save the Experiment. 4 Keep the filename Tutorial.exp and navigate to: C:\ FCAP Assays NOTICE You may need to create this folder. Alternatively, you may choose to save your Experiment in a different location. 42 FCAP Array Software Tutorial Rev. A

43 5 Click Save. The file path of the saved Experiment is listed in the main window title bar Rev. A FCAP Array Software Tutorial 43

44 Exporting the Experiment File Set Up Instrument Transfer Template Create FCAP Experiment Export Experiment Import Experiment Acquire Data Analyze Data 1 If needed, click the Plate Layout tab to display the Plate Layout view. 2 Click Export Experiment. The Experiment Export Wizard opens in the Select Template view. 3 Select the BD CBA Flex Set Setup mmddyy.xml template that you created in Saving the Instrument Setup Experiment as a Template on page FCAP Array Software Tutorial Rev. A

45 4 Click Next. The wizard advances to the Set Stopping Rule view. 5 Set the number of events per Bead to be acquired. The recommended value is 300 events per Bead. NOTICE The total number of events acquired is determined by the software, by multiplying the events per Bead value by the number of Beads in the Experiment. For example, when using the recommended 300 events per Bead with the 10-plex of this Experiment: = 3000 total events are acquired. In this case, exactly 3000 total events are acquired, with approximately 300 events acquired for each Bead. The exact number of events acquired for a given bead is dependent on both sampling statistics and the quality of the sample preparation Rev. A FCAP Array Software Tutorial 45

46 6 Click Next. The wizard advances to the Select Folder view. 7 Click Browse. A Browse for Folder dialog is displayed. NOTICE It is okay to choose a folder location different than the one specified below. If so, remember that location for Importing Experiment File into BD FACSArray Software on page Navigate to C:\Program Files\BD FACSArray System Software and create a new folder there named FCAP Array Files. 9 Click OK. 46 FCAP Array Software Tutorial Rev. A

47 10 Click Finish. The wizard advances to the Complete view. 11 Click Close. A new folder is created in the FCAP Array Files folder to hold the exported XML Experiment file. The new folder name (Tutorial) is the name of the FCAP Array software Experiment. 12 Exit from FCAP Array software Rev. A FCAP Array Software Tutorial 47

48 Importing Experiment File into BD FACSArray Software Set Up Instrument Transfer Template Create FCAP Experiment Export Experiment Import Experiment Acquire Data Analyze Data 1 Start up the BD FACSArray bioanalyzer. Refer to the BD FACSArray System User s Guide for the startup procedure. 2 Launch BD FACSArray software. The default user login is Administrator and the password is welcome. 3 In BD FACSArray system software, select File > Import Experiments. 4 Navigate to the C:\Program Files\BD FACSArray System Software\FCAP Array Files\Tutorial folder. Do not open the Tutorial folder. Select the folder and click Import. The imported Experiment, named Tutorial, is added to the database of BD FACSArray software and appears in the Browser. 48 FCAP Array Software Tutorial Rev. A

49 5 Locate the imported Experiment in the Browser of BD FACSArray software, and double-click the Experiment icon to open the Experiment Rev. A FCAP Array Software Tutorial 49

50 Acquiring Data Set Up Instrument Transfer Template Create FCAP Experiment Export Experiment Import Experiment Acquire Data Analyze Data Sample Preparation Before acquiring data, you must prepare a plate with the assay samples. For detailed information on preparing assay samples for a plate, see your BD CBA Human Soluble Protein Master Buffer Kit Manual. The following tables contain reference information for your assay sample preparation. Plex Analytes Human IL-2 Human IL-4 Human IL-5 Human IL-6 Human IL-8 Human IL-1β Human IL-10 Human TNF Human IL-12p70 Human IFN-γ Well Sample Concentration A1 Std A2 Std A3 Std A4 Std A5 Std A6 Std A7 Std A8 Std A9 Std A10 Std B1 Tutorial001 unknown B2 Tutorial002 unknown 50 FCAP Array Software Tutorial Rev. A

51 NOTICE If you are performing this tutorial in a class setting, your instructor will provide the test samples Tutorial001 and Tutorial002. If you are following the tutorial on your own, create test samples with known concentrations, so that you can verify your analysis results. Sample Acquisition 1 Ensure that the sample layout of the FACSArray Experiment reflects the layout in the prepared plate. 2 Click the Setup tool ( ) in the application toolbar to display the Setup workspace. 3 Place the assay plate (containing the standard and test samples) in the loading tray of the BD FACSArray bioanalyzer so that well A1 is aligned over the A1 position label on the plate holder. 4 Click Load to load the prepared plate (Plate control group of the Plate Editor) Rev. A FCAP Array Software Tutorial 51

52 5 Click Acquire to start acquisition of all the wells. 6 When acquisition completes, click Unload. 7 Unload the assay plate from the instrument. 8 Click the Prepare tool ( ) in the application toolbar to display the Prepare workspace. 9 In the Browser, select the Tutorial Experiment. 52 FCAP Array Software Tutorial Rev. A

53 10 Export the FCS 2.0 list-mode files from this Experiment: a Select File > Export > FCS. The Export Parameter dialog is displayed. b c Select FCS 2.0 as the file version to export. Click OK. The Export Parameter dialog is displayed. d Click Save to save the FCS files at the default location D:\BD Export\FCS. The Export FCS file(s) window indicates FCS file export progress. 11 Shut down the BD FACSArray bioanalyzer. See the shutdown procedure in the BD FACSArray System User s Guide Rev. A FCAP Array Software Tutorial 53

54 Analyzing Data Set Up Instrument Transfer Template Create FCAP Experiment Export Experiment Import Experiment Acquire Data Analyze Data Analyzing Data a Opening the Saved Experiment b Performing File Assignment c Assigning Beads to Clusters d Starting the Analysis e Printing a Report f Inspecting the Standard Curves g Reviewing the Raw Data h Saving the Plex (Optional) The FCAP Array software bead assay data analysis includes cluster identifications, MFI value calculations, curve fittings, and individual analyte concentration determinations. Opening the Saved Experiment If needed, launch FCAP Array software and open the Experiment you created in the section Creating an Experiment in FCAP Array Software on page Launch FCAP Array software. 2 Select File > Open existing experiment. An Open dialog is displayed. 3 Navigate to the Tutorial.exp Experiment file you created in step 5 on page 43: C:\ FCAP Assays\ Tutorial.exp NOTICE If you saved the Experiment document in an alternate location, you should navigate to that location instead. 4 Click Open. Performing File Assignment The FCAP Array software File Assignment view is used to assign FCS 2.0 data files to corresponding Experiment samples. 1 Click the File Assignment tab to display the File Assignment view. 54 FCAP Array Software Tutorial Rev. A

55 NOTICE If the dialog shown in Figure 1-8 is displayed, click OK and continue. Figure 1-8 File assignment diagnostic dialog The left side of the File Assignment view lists the Experiment samples. 2 Use the Instrument name pull-down to select BD FACSArray. 3 Enter your BD FACSArray bioanalyzer serial number in the Serial number field. Tip The actual serial number is not required for bead assay analysis. If you do not know your BD FACSArray bioanalyzer serial number, enter unknown. 4 Click the Folder button. The Select Data File dialog is displayed Rev. A FCAP Array Software Tutorial 55

56 5 Navigate to the folder containing the FCS 2.0 data files for the Experiment: D:\BD Export\FCS\Tutorial\Plate_001 6 Highlight any one of the FCS files and click Select. The FCS files are loaded and the right side of the File Assignment view lists the FCS files found in the folder you selected. The software attempts an automatic file assignment. 56 FCAP Array Software Tutorial Rev. A

57 7 If the dialog shown in Figure 1-8 on page 55 is displayed, click OK and continue with manual file assignment: a In the sample list (left side of the File Assignment view), select the first row. With the Data files list (right side) select the corresponding FCS data file name. One row in each list is highlighted. b Click. The view changes to indicate that the selected FCS data file is assigned to the corresponding sample name. Repeat steps a and b above with subsequent rows of the sample list until all files are assigned Rev. A FCAP Array Software Tutorial 57

58 When the file assignment is complete: The left side of the view indicates FCS file assignments. The files on the right side of the File Assignment view are marked to denote that they have been assigned. The Start analyzing this experiment toolbar icon is active. 58 FCAP Array Software Tutorial Rev. A

59 Assigning Beads to Clusters 1 To begin the analysis, click the Start analyzing this experiment icon. Bead-cluster assignments were skipped when the Experiment was created, so a dialog indicates that cluster assignment must be performed prior to data analysis. 2 Click OK to continue with the Bead-cluster assignment. The Clustering Parameters view of the Plex Modification Wizard is displayed. 3 Click Load Data File and select an Experiment data file as in the previous section (see steps 5 and 6 on page 56) Rev. A FCAP Array Software Tutorial 59

60 4 Use the Instrument name drop-down menu to select BD FACSArray. The remaining fields are automatically populated with default values for the BD FACSArray bioanalyzer. 5 Verify that your settings match those shown in the figure, and that the message indicates that all 10 of the plex clusters were found. 6 Click Next to advance to the Analyte Assignment view. 7 Assign Beads to clusters: a Click the first line of the Bead-cluster list (Cluster ID = 1). The Bead information is highlighted. b Double-click the left-most cluster in the top row of clusters (labeled 1 in Figure 1-9 on page 61) in the dot plot to assign the selected Bead to that cluster. The Cluster ID of the Bead appears near the cluster, indicating that the Bead has been assigned accordingly. The number of collected events for that cluster is also displayed in the list. The Bead-cluster list selection advances to the next line (Figure 1-9 on page 61). c As in step b, assign the remaining Beads to their corresponding clusters. The Bead ID location codes indicate relative cluster positions. Use the cluster position map on the right side of Figure 1-10 on page 61 to determine the Bead-cluster assignments. The correct completed assignment is shown on the left side of Figure 1-10 on page 61. If you make a mistake, click Clear Assignment to restart. 60 FCAP Array Software Tutorial Rev. A

61 d When the Bead-cluster assignment is complete, click Finish. Figure 1-9 Assigning a Bead to its cluster Figure 1-10 Completed Bead-cluster assignment NOTICE Correct assignment of Beads to clusters is necessary for proper reporting of the analysis results Rev. A FCAP Array Software Tutorial 61

62 Starting the Analysis 1 To start the analysis, click the Start analyzing this experiment icon: Since the Bead-cluster assignments are complete for each Plex in the Experiment, data analysis begins. When the analysis is complete, the Analysis Messages dialog is displayed. 2 Click OK to dismiss the dialog. Three new tabs are now displayed in the main window: Report Printout Standard Curves Raw Data 3 Before reviewing the analysis results, safeguard the new analysis data by saving the changes to your existing Experiment by clicking the Save this experiment icon: 62 FCAP Array Software Tutorial Rev. A

63 Printing a Report In the Report view, you view, format, and print an analysis report. 1 Click the Report Printout tab to display the Report Printout view (Figure 1-11 on page 63). Figure 1-11 Report view The Full report is displayed in the Report view. By default, it consists of: Experiment identifiers Sample/FCS file table Statistics from the standard samples and the curve fit Standard curve plots Color-coded charts of test sample concentrations (without dilution factor applied) Statistics from the test samples Rev. A FCAP Array Software Tutorial 63

64 2 Scroll down through the Full report. You can modify the contents and formatting of the Full report, or create additional custom report templates (see FCAP Array Software User s Guide). 3 Click Page Setup... to specify page information, choose a printer, and select printer properties. 4 Click Print to print the Full report. Inspecting the Standard Curves In the Standard Curves view, you can inspect and print standard curve plots and apply a new curve fitting model to the data. 1 Click the Standard Curves tab to display the Standard Curves view. The standard curve for the Human IL-2 analyte is displayed. 2 Change the selection in the Analytes scroll box to view the Human IL-4 standard curve. 64 FCAP Array Software Tutorial Rev. A

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66 3 Print one or more of the standard curve plots. a b Select File > Page Setup to specify page information, choose a printer, and select printer properties. Click Print. The Select Clusters to Print dialog is displayed. c d Choose one or more standard curves by marking the checkboxes next to the analytes. Click OK. The standard curve plots are printed. 66 FCAP Array Software Tutorial Rev. A

67 Reviewing the Raw Data In the Raw Data view, you inspect analysis statistics and plots. 1 Click the Raw Data tab to display the Raw Data view. Median (MFI) statistics are shown for every analyte of every sample Rev. A FCAP Array Software Tutorial 67

68 2 Click the Event Num, SD, %CV, Sample CC (calculated concentration), and Final CC buttons to display those statistics. 3 Click Histogram to create a plot window. 4 Click Statistics to create a statistics window. 5 Click on cells of the table and note the changing contents of the plot and statistics windows. 68 FCAP Array Software Tutorial Rev. A

69 6 (Optional) To export a comma delimited raw data file (.csv): a Click Export. The Raw Data Export dialog is displayed. b c d e f Choose to export Samples and Statistics by marking their checkboxes. Select Samples in rows, analytes in columns. Click Continue. Specify a file name and location in the Specify Export File dialog that follows, and click Export. Open the file you created in a.csv compatible spreadsheet (example: Microsoft Excel) Rev. A FCAP Array Software Tutorial 69

70 Analysis Results Fill in the table by using the Raw Data view to obtain the test sample cytokine concentrations (Final CC). Cytokine Tutorial001 Final Concentration Tutorial002 Human IL-2 Human IL-4 Human IL-5 Human IL-6 Human IL-8 Human IL-1β Human IL-10 Human TNF Human IL-12p70 Human INF-γ 70 FCAP Array Software Tutorial Rev. A

71 Saving the Plex (Optional) You can save and reuse your Experiment Plex in future Experiments. This simplifies both the Experiment creation process and Bead-cluster assignments. NOTICE A plex can be saved only if it has a completed Bead-cluster assignment. 1 Click the Experiment tab to display the Experiment view. 2 In the Experiment view browser, right-click the Plex, and select Save as... from the context-sensitive menu. 3 In the Save Plex dialog, enter a plex name, and click OK. The Experiment view browser is updated with the saved Plex name Rev. A FCAP Array Software Tutorial 71

72 Finalizing the Experiment (Optional) In this section, you finalize the Experiment to prevent additional modifications. When analysis and review are complete, you can prevent additional modifications to an Experiment by finalizing it. The Report generated and printed from a finalized Experiment is also locked. However, the format and the reported data selection of a locked Report can still be modified. 1 Click the Finalize icon in the application toolbar: The Signing experiment dialog is displayed. 2 Choose a user name with signing authorization from the Login name drop-down menu. If you have an authorized account choose your own user name. Otherwise, choose Administrator. 3 Enter the password (default password for the Administrator account is welcome). 4 Click OK. When an Experiment is finalized, the Finalize icon in the toolbar is replaced with a Lock icon to signify the locked status of this Experiment. 5 The analysis is complete. You can exit FCAP Array software. 72 FCAP Array Software Tutorial Rev. A