BD LSR Fortessa How To Use - IGC - Facilities...

Flow Cytometry Service BD LSR Fortessa SORP – How to Use Guide

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Contents

Section 1 Instrument Startup Page 2

Section 2 Switch from acquisition in plate to acquisition in tubes Page 3

Section 3 FACS DiVa setup for tubes & automatic compensation Page 4

Section 4 FACS DiVa setup for plates Page 7

Section 5 Cleaning procedures, data export & cytometer shutdown Page 9

Section 6 Cleaning procedures after acquisition in plate 1

Section 7 Troubleshooting Page 13

If you have an emergency with your instrument, please contact the Flow Cytometry Staff:

From 9AM to 6PM: 214 407 951

After 6PM and weekends: 917 262 274

WARNING

Ensure your cells/particles are resuspended in PBS containing NO MORE than 2% serum.


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Section 1: Instrument Startup

1 The instrument is: OFF (go to step 2); ON (go to step 4).

2 Turn ON the cytometer in the power extension switch. Confirm the computer was OFF before you do this. Otherwise, switch OFF the computer first. Be aware that the lasers take about 15 minutes to warm up and stabilize.

3 Turn ON the computer and screens.

4 Sign in to your Agendo account. Ensure the indication “Agendo connected” was displayed in the dialog window before you sign in.

5 Launch FACS DiVa software and log in as Administrator (no password needed).

6 Confirm that the PBS tank is full. Otherwise, fill it up with PBS solution taken from the tank labeled “PBS Fortessa” and report the situation through Agendo, using the “Report incident” option.

7 Wait for the indication “Cytometer connected” on the bottom of the Cytometer panel.

8 In the FACS DiVa software select “Keep Current Settings” if you are using bacteria or yeast. If you are using, cells select “Use CST Settings”.

9 If you will make your sample acquisition in FACS tubes, please follow the instructions to switch from acquisition in plate to acquisition in tubes provided in Section 2. If your acquisitions will be performed in plate, go directly to Section 4.

IMPORTANT NOTE: The dead volume of BD LSR Fortessa SORP is around 70 µL. For this reason, we recommend to resuspend your samples in a minimal volume of 200 µL. It is strictly forbidden to remove the outer needle of the SIP1. If you believe you need to do it, please contact the facility staff.

1 SIP – Sample Injection Port: the tube/needle that goes inside the FACS tube and through which the sample is aspirated.


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Section 2: Switch from acquisition in plate to acquisition in tubes The standard configuration of the cytometer is to acquire samples in 96-well plates using the High Throughput Sampler (HTS). If you need to switch the configuration to acquire your samples in FACS tubes, please follow these steps:

1 Unscrew the sample coupler until you are able to remove it from the SIP.

2 Leave the sample coupler on the round, white-sponge in the back of the HTS.

3 Unscrew the SIP protector and remove it from the SIP. Ensure the O-ring sitting on the top of the SIP stays in place.

4 Install the outer needle of the SIP that is stored in the 15-mL Falcon tube immersed in ethanol.

5 Leave in the same Falcon tube the SIP protector during your acquisition.

6 On the right side of the cytometer, move the black switch from plate to tube mode.

7 You are ready to start acquiring your samples in FACS tubes. Go to Section 3 for further guidance.

After acquiring samples in tubes and performing the cleaning procedures (see Section 5), please set the configuration of the cytometer to plate acquisition following the next steps:

1 On the right side of the cytometer, move the black switch from tube to plate mode.

2 Unscrew the outer needle of the SIP and remove it carefully to prevent damage of the SIP. Ensure the O-ring sitting on the top of the SIP stays in place.

3 Store the outer needle of the SIP in the 15-mL Falcon tube containing ethanol after taking out the SIP protector.

4 Attach the SIP protector.

5 Install the sample coupler by screwing the upper part until it is fixed to the SIP.

6 Confirm the sample protector is well installed and there is no PBS leaking.

7 You are ready to start acquiring your samples using the HTS.


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Section 3: FACS DiVa setup for tubes & automatic compensation

1 Confirm that all panels are visible: Browser, Cytometer, Inspector, Acquisition Dashboard & Global Worksheet.

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Informative Those windows have the following functions: Browser: to create and manage experiments, specimens and tubes for sample acquisition and recording. Instrument settings may also be copied/pasted, liked/unlinked in this area. Cytometer: to provide information about the cytometer and allow the adjustment of several parameters, such as voltages, compensation, laser delay, thresholds, etc. Inspector: to provide information about and allow the modification of the elements previously selected (e.g. if we select a dotplot, Inspector shows the options to activate the bi-exponential display, to show a grid, to change the background color, etc.; if we select a tube in the Browser panel, Inspector allows to change the tube name, etc. Acquisition Dashboard: controls the sample acquisition and allows to select the number of events shown in the graphs and to be recorded. Global Worksheet: panel where the dotplots and histograms are displayed. It has 2 modes: Global Worksheet (works as a ‘template’ for all tubes and applies every modification to all tubes) and Normal Sheet, which allows individual analysis for each tube, meaning that any change (in a gate, for instance) made in a particular tube will not affect the analysis of the other tubes. To alternate between these two modes one should click in the first button on the top left of this panel.

3 At the top of the Browser panel, create a New Experiment (click the icon with a book).


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4 Create a New Specimen (click the icon with a syringe).

5 Select Tube_001 (inside the syringe) by clicking in the green arrow.

6 Do 3 PRIMEs with the SIP2 arm open and no tube installed. Then set the cytometer to RUN.

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Acquire and record dH2O for 3 minutes in HIGH (name it ‘Initial’). Check in the FSC/SSC dotplot if any events appear. If you get too many events per second and/or falling into your gates, replace the dH2O tube by a new one prepared by you. If the problem persists, perform the cleaning procedure as indicated in Section 5 and report the situation through Agendo, using the “Report incident” option.

8 If the machine is clean, you can start acquiring your samples (Step 21) or perform Automatic Compensation (Step 9).

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To perform automatic compensation, do to Experiment Menu à Compensation Setup à Create Compensation Controls. This will open a window entitled Create Compensation Tubes. Confirm the detectors displayed are the ones you will need. Otherwise, delete the ones you won’t use. If you do not have an Unstained Control, unselect the option “Add Separated Unstained Control”. Click OK.

10 In the Acquisition Dashboard panel choose to display 500 events.

11 In the Browser panel, scroll down the tubes hidden under the Compensation Control Specimen and select the unstained tube by clicking in the green arrow.

12 In the Worksheet window, select all histograms with the aid of the Shift key. Then go to the Inspector panel and select “grid” to visualize four quadrants in all histograms.

13 Install the unstained control sample in the cytometer and set the speed to LOW. Start acquisition in the Acquisition Dashboard.

2 SIP – Sample Injection Port: the tube/needle that goes inside the FACS tube and through which the sample is aspirated.


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14 In the Cytometer panel, in the Parameters tab, adjust the voltages of FSC and SSC in order to roughly place your cells of interest at the center of the dotplot.

15 Adjust P1 gate around your population of interest. Then, right click in the mouse and select “Apply to All Compensation Controls”.

16 In the Cytometer panel, in the Parameters tab, adjust the voltages of each fluorescence detector so the negative cells sit in the left quadrant of the histograms, touching the right border. Before recording each control, confirm that each fluorochrome is the brightest in its respective channel.

17 Record the unstained tube and click Next Tube.

18 Install the first Single Staining Control tube in the cytometer. Record all the events and adjust the P2 region around the positive population. Click Next Tube.

19 Repeat the previous step for all Single Staining Controls.

20 Go to Experiment Menu à Compensation Setup à Calculate Compensation and select Link & Save.

21 In the Browser panel select Tube_001 by clicking the green arrow.

22 In the Cytometer panel open the Parameters tab and select the detectors/fluorochromes that you will not use with the aid of the shift key. Delete them by pressing the button at the bottom. Warning: keep FSC-PMT and SSC-violet since these are the optimized detectors for FSC and SSC.

23 In the Worksheet window choose between the option of Global Worksheet or Normal Worksheet according to your purpose. We recommend using Global Worksheet for acquisition.

24 In the Cytometer panel select “W” (in addition to “A”) in the FSC parameter. This will be useful in case you need to exclude doublets.

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In the Worksheet window create a dotplot FSC-A versus SSC-A to select your primary population – P1; and a dotplot FSC-A versus FSC-W to exclude doublets – P2. To create a gate inside another, right click in the mouse while touching a dotplot and select Population Hierarchy, which will open a new window with that information. In that window select the region you would like to have as parent gate and create a new gate. Add all the dotplots and histograms you will need for your analysis and draw the draft of the gates in each one of them.


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26 In the Experiment menu select “Experiment Layout” and write the name of the molecules you are analyzing in the corresponding fluorochrome/channel.

27 In the Acquisition Dashboard panel select the number of events to visualize and to record, as well as the gate inside which the events should be counted and recorded.

28 You are set to start your acquisition and record your samples. Be aware of the volume aspirated during acquisition to avoid air to enter the cytometer.


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Section 4: FACS DiVa setup for plates

1 Confirm that all panels are visible: Browser, Cytometer, Inspector, Acquisition Dashboard & Global Worksheet.

2

Informative Those windows have the following functions: Browser: to create and manage experiments, specimens and tubes for sample acquisition and recording. Instrument settings may also be copied/pasted, liked/unlinked in this area. Cytometer: to provide information about the cytometer and allow the adjustment of several parameters, such as voltages, compensation, laser delay, thresholds, etc. Inspector: to provide information about and allow the modification of the elements previously selected (e.g. if we select a dotplot, Inspector shows the options to activate the bi-exponential display, to show a grid, to change the background color, etc.; if we select a tube in the Browser panel, Inspector allows to change the tube name, etc. Acquisition Dashboard: controls the sample acquisition and allows to select the number of events shown in the graphs and to be recorded. Plate: to select wells in the plate, add specimens and tubes, and define HTS settings of acquisition. Global Worksheet: panel where the dotplots and histograms are displayed. It has 2 modes: Global Worksheet (works as a ‘template’ for all tubes and applies every modification to all tubes) and Normal Sheet, which allows individual analysis for each tube, meaning that any change (in a gate, for instance) made in a particular tube will not affect the analysis of the other tubes. To alternate between these two modes one should click in the first button on the top left of this panel.


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3 At the top of the Browser panel, create a New Experiment (click the icon with a book).

4 Create a New Plate (click the blue icon with a plate).

5 If you had switched on the HTS with the software running you must reinitialize the HTS. Go to HTS menu → reinitialize.

6 Go to HTS menu → select Prime 3 times. Ensure there are no bubbles in the system.

7 Select the wells you want to analyze and add a New Specimen in the Plate Window.

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Define the loader settings: • Sample Flow Rate (µl/sec): is the speed the syringe injects sample on the cytometer in μL/second. You should not have more than 10.000 evts/s. • Sample Volume: is the volume of sample aspirated from each well for acquisition. During acquisition in standard mode, HTS aspirates the selected sample volume plus an additional 20 μL from the well. Ensure each well contains sufficient sample for the entered volume plus the dead volume. Insufficient volume can introduce air bubbles into the system. BD recommends that you prepare your plate with a minimum of 250 μL of sample/well for a 96-well plate in standard mode, 100 μL/well for a 96-well plate in high throughput mode, and 50 μL/well for a 384-well plate (either mode). • Mixing Volume: is the volume of sample aspirated and dispensed during mixing. Ensure each well contains sufficient sample for mixing. BD recommends a mixing volume that is one-half the available volume. • Mixing Speed: is the speed the syringe aspirates and dispenses the sample to the well during mixing. This parameter is specified by the user according to the cell type and experiment layout. • Number of Mixes: is the number of mixing cycles performed before a sample is aspirated. is specified by the user according to the cell type and experiment layout. • Wash Volume: is the volume of sheath fluid dispensed for rinsing between wells. is specified by the user according to the cell type and experiment layout. IMPORTANT: if you are using PI, please use the maximum wash volume (800 μl).


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Acquire and record a first well with dH2O (name it ‘Initial’). Check in the FSC/SSC dotplot if any events appear. If you get too many events per second and/or falling into your gates, replace the dH2O and repeat acquisition. If the problem persists, perform the cleaning procedure as indicated in Section 6 and report the situation through Agendo, using the “Report incident” option.

10 You are ready to start running your samples.

IMPORTANT NOTE: Please do not include more than one plate in the same experiment to avoid database corruption. Save each of your acquired plates in separated experiments. Thank you.


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Section 5: Cleaning procedures, data export & cytometer shutdown* *For cleaning procedures using the HTS, please go to section 6.

1 If you have used propidium iodide (PI) or other viability dyes go to step 2. If you did not use PI or other viability dye staining in your experiment, go to step 8.

2 If you have an extra sample with cells not labelled with PI at relatively high concentration, go to step 3. If you don’t, go to step 4.

3 Perform a 3-minute acquisition of your PI-unlabeled concentrated cells in HIGH. Record this acquisition (name it ‘Unstained to Clean PI’). At the end go to step 8.

4 Install a tube of bleach with the SIP arm open for 1 minute.

5 Close the SIP arm and aspirate bleach in Super HIGH for 5 minutes. Record this acquisition (name it ‘Bleach’).

6 Install a tube of detergent with the SIP arm open for 1 minute.

7 Close the SIP arm and aspirate detergent in Super HIGH for 10 minutes. Record this acquisition (name it ‘Detergent’). At the end, go to step 12.

8 Install a tube of bleach with the SIP arm open for 1 minute.

9 Close the SIP arm and aspirate bleach in Super HIGH for 5 minutes. Record this acquisition (name it ‘Bleach’).

10 Install a tube of detergent with the SIP arm open for 1 minute.

11 Close the SIP arm and aspirate detergent in Super HIGH for 5 minutes. Record this acquisition (name it ‘Detergent’).

12 Install a tube of dH2O with the SIP arm open for 1 minute.

13 Close the SIP arm and aspirate dH2O in Super HIGH for 5 minutes. Record this acquisition (name it ‘H2O’).

14 Set the cytometer in Standby. Go to Section 2 and change the cytometer mode to acquisition in plate.

15 Refill the sheath tank with PBS from the “PBS Fortessa” container.

16 Export your Experiment from FACS DiVa by right clicking on the Experiment name with the mouse and selecting Export à Experiment. Select the local destination folder for temporarily saving your files (usually there is a folder in the Desktop).

17 DELETE your experiment from DiVa. If you wish to keep your settings and page analysis, copy the Experiment and paste it in DiVa without data.


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18 Copy the exported files to your personal directory in the remote server by launching Fileszilla from the Start Menu. You'll be prompted for your username and password and after that just need to select the folder you want to access and drag your local files into the remote folder.

19 After transferring your data to the remote server, delete the files from the local folder.

20 Quit DiVa (Menu File → Quit).

21 Check if there is a booking after you in the next 3 hours. If YES, go to step 22. If NO, go to step 23.

22 Leave the cytometer in Standby with dH2O on the SIP and log off from Agendo. Remove your gloves, tubes and other belongings and clean up any trash. You are done.

23 Switch OFF the power extension connected to Fortessa.

24 Log off from Agendo and turn the computer off. Remove your gloves, tubes and other belongings and clean up any trash. You are done.


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Section 6: Cleaning procedures after acquisition in plate

1 If you have not used PI, go to step 2. If you have included PI in your staining, go to step 4.

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Record the acquisition of one line in a 96-well plate as follows: - 3 wells with 300 µL bleach named ‘Bleach’ - 3 wells with 300 µL dH2O named ‘H2O’ - 3 wells with 300 µL detergent named ‘Detergent’ - 3 wells with 300 µL dH2O named ‘H2O’

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Run the selected wells with the following settings:

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If you have used PI or other viability dye, select one of the next options: OPTION 1: Acquire 3 wells of very concentrated unstained cells (named ‘Unstained to clean PI’) and then run the cleaning protocol described in steps 2&3. OPTION 2: Perform the cleaning protocol described in steps 2&3, but add 3 extra wells with detergent.

5 Set the cytometer in Standby and quit DiVa (Menu File → Quit).

6 Refill the sheath tank with PBS from the “PBS Fortessa” container.

7 Export your Experiment from FACS DiVa by right clicking on the Experiment name with the mouse and selecting Export à Experiment. Select the local destination folder for temporarily saving your files (usually there is a folder in the Desktop).

8 DELETE your experiment from DiVa. If you wish to keep your settings and page analysis, copy the Experiment and paste it in DiVa without data.

9 Copy the exported files to your personal directory in the remote server by launching Fileszilla from the Start Menu. You'll be prompted for your username and password and after that just need to select the folder you want to access and drag your local files into the remote folder.

10 After transferring your data to the remote server, delete the files from the local folder.

11 Check if there is a user reserved after you in the next 3 hours. If YES, go to step 12. If NO, go to step 13.


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12 Leave the cytometer in Standby and log off from Agendo. Remove your gloves, tubes and other belongings and clean up any trash. You are done.

13 Switch OFF the power extension connected to Fortessa.

14 Log off from Agendo and turn the computer off. Remove your gloves, tubes and other belongings and clean up any trash. You are done.

BD LSR Fortessa How To Use - IGC - Facilities...

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