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FACScan training instruction by Flow Cytometry Core Facility of Mount Sinai Shared Facility Last updated 3/11/2010 Notes: i) Any problems with the instrument should be reported by email to siu-hong.ho@mssm.edu, or italas.george@mssm.edu and a note left with the instrument to inform the next user. ii) Two kinds of tube are commonly used in the lab, polypropylene and polystyrene tube (more transparent). The machine can only take polystyrene tube. A) Start up Procedure

1) Check the fluidic system, it composed of the buffer and waste tank.

2) Sheath tank is at least 1/3 full. Otherwise, release the pressure valve by flipping the “Vent valve”, unscrew the assembly on the sheath tank. Get the sheath tank with sheath fluid on top of the machine and screw the assembly back to the sheath tank. For Calibur, you may need to remove the black metal cover on top of the sheath tank also.

Sheath/waste Sheath/waste

Sheath Waste Sheath Waste

Calibur ScanExtra sheath/empty waste tank

Extra sheath/empty waste tank

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3) You may as well change the waste tank in case you change the sheath tank. Unscrew the assembly on the waste tank, get an empty waste tank on top of the machine and screw the assembly back to the waste tank. 4) Flip the “Vent value” back to the pressurized position. THIS IS THE STEP MOST EASIEST TO FORGET. 5) Switch on the cytometer. The cytometer must be on before the computer. If you found that the computer is on while the cytometer is off, turn off the computer before you turn on the cytometers. For the Scan, the status of the machine is shown as “Not Ready”. When it changes from “Not Ready” to “Standby”, the machine is ready to take sample. For the Calibur, it’s already “Standby”, but we suggest you to wait at least 5 minutes for the machine to warm up.

6) Switch on the computer. B) Controlling the hardware of the cytometer

1) In the front panel, you will find the fluid control and you will find a button and a switch. The button controls “Hi” and “Lo”. The machine is taking up the sample by pressurizing the sample tube, the content of the sample tube would be pushed into the machine once it got pressurized. “Hi” means high pressure applied and the sample would be taken up faster; “Lo” means low pressure applied and the sample would be taken up slower. In Calibur, there is “Med”, the extent of pressurization of “Med” is in between “Lo” and “Hi”.

Calibur

Scan

Metal cover

Pressure valve

Calibur Scan

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2) The switch has five options: “Standby”, “Run”, “Drain”, “Fill" and “Backflush”. “Standy” means the machine is not pressurizing the sample; “Run” means the machine is pressurizing the sample. You may ignore the “Backflush” function. 3) During the data acquisition, if you found your population is moving up and down and left and right crazy, there may be air bubble trapped in the flow cell (analytical unit inside the machine). Then you may use the “Drain” and “Fill” option in the Scan. Flip to “Drain” for 30 seconds then to “Fill” for the other 30 seconds”. The fluid will be drained out from the flow cell and you would see air bubble coming out into the sample tube when the switch is in “Drain”. Then the flow cell would be filled again by the buffer when the switch is in “Fill”. For the Calibur, “Prime” button does both drain and fill. 4) Changing from sample to sample: a) while the machine is still in “Run”, hold the sample tube with one hand; b) flip the arm below the sample tube either to the left or right with the other hand; c) take the sample tube out; d) put the other sample tube in; e) flip the arm back to the middle. (Notes: i) While the machine is still in “Run”, the tube is pressurized. Holding the tube is very important, otherwise, there is chance the tube would come out like a bullet and the content will spill into body. ii) Once the arm is flipped to either left or right, the tube is not pressurized. However, there is a pump activated to take the dripping from the machine, it also takes your sample. Therefore, once you flip the arm to one side, take the sample tube out as soon as possible to prevent the machine from taking up your sample. You may need it for re-run) 5) Before you put in any sample, clean the machine with 10% bleach and water. Fill the tube with “bleach” label with 1ml bleach from the spray bottle (never fill more than 1ml, otherwise the sealing part would be eroded), put into the sample dock, flip the fluid control to “Hi” and “Run” for 3 minutes; then flip the arm to either left or right for 10 seconds to clean the waste line. Repeat the same by using water. Then flip the fluid control to “Lo” and “Standby”. C) Controlling the software of the cytometer

1) Log on to the computer with your PI’s name and the last four digits of the grant number.

Calibur Scan

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2) FACStationGF BD Applications CellQuest Pro Folder “CellQuest Pro”.

3) When you open “CellQuest”, you will get a blank template, we will get back here later.

4) You will spend a little while on the “Acquire” menu, you will go through “Acquistion & Storage”, “Parameter Description”, “Counters” and “Connect to Cytometer”

Template

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5) You first go to “Acquisition & Storage”, a window will pop up. The only thing you need to worry about is the “Acquisition will stop when”.

Enter a number there and the program will save this number of events for each sample. The default is 10,000, this number is good for may be 90% of the experiment. However, if you are analyzing rare population, you may need to increase this number. 6) Then go to “Parameter Description”, a window will pop up again.

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You will go through from (i) to (ix). i) Directory means where to store your data, click change and probably create a folder in the desktop. ii) “File” specifies how you name individual sample.

The default is “Data” and “1” meaning the first file would be named as Data.001 and the second file would be Data.002 and so on. You may also click the pull down manual of the “File Name Prefix”, from the manual, you can change from “Custom Prefix” to “Sample ID” followed by clicking “OK”

In this case, what you type in the “Sample ID” field would be your file name. For example, if you type in “Control” for your first sample, then your first file name would be “Control.001”; then you type in “Positive” for the second sample, the second file name would be “Positive.002”. The name would

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iv v vii vi viii

ix

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be changed according to what you type in and the number that follows would increase by 1. iii) “Sample ID” is mentioned in (ii). It tells the information of the sample. iv) P1 to P5 and P7 are the parameters the machine can measure, P1 is Parameter 1 and P2 is Parameter 2 and so on. When you click the pull down manual of P1, you can choose either one of the option, then that option would be the name of the axis in the plot (the explanation of the plot will be covered below) or you can type whatever name which you think more fit to your experiment. P1 is “forward scatter”, the meaning of P1 is illustrated by the slides attached at the end. ONLY Calibur has P7.

v) P2 is “side scatter”, the meaning of P2 is illustrated by the slides attached at the end. PBMC is one of the best examples to show how the population could be separated by forward scatter and side scatter.

vi to ix) P3 to P7 are channels for measuring fluorescence with different wavelength. They are named as FL-1, FL2, FL-3 and FL4, respectively, for the time being. You may name the channels according to your actually experiment, e.g. CD3-FITC, CD4-PE, CD8-PerCP, etc. The meaning of the fluorescent channels is explained in the slides attached at the end. For Calibur only You also need to click the “Four Color” in the “Detector/Amps” to activate the P7 parameter. How to open this window would be covered in the following session.

Pull down manual

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7) You are still in the “Acquire” manual. You’ve gone through the “Acquisition & Storage” and “Parameter Description”. Now, click the “Counter”, you will see:

You don’t need to do anything for this window. It just tells you how many cells the machine is analyzing per second and how many cells the machine has saved so far. This window is for information purpose only. 8) Then still under the “Acquire” manual, click “Connect to Cytometer”. A new windows pops out and we will get into that later.

9) Then go to the “Cytometer” manual and click the “Detector/Amps” option.

A new window pops out, which allows you to control the sensitivity of different channels. i.e. FL1, FL2 and FL3. And now you know how to open the window with “Four Color”. For the P1, i.e. Forward Scatter, you have the “Voltage” and “Amp Gain” to control the sensitivity of forward scatter sensor. “Voltage” is like the coarse adjustment of the microscope and “Amp Gain” is like the fine adjustment of the microscope. For all other parameter, both coarse and fine adjustment are in the “Voltage”.

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When you click the pull down manual of “Voltage” of P1, i.e. Forward Scatter, you will have “E00”, “E01”, “E02”, “E03” and “E-1” options (not shown here). “E00” means the signal is what it is, there is no manipulation of the signal; “E01” means the signal gets amplified by 101 time, i.e. 10 time; “E02” means 102 time, i.e. 100 time; “E03” means 103 time, i.e. 1000 time; “E-1” means 10-1 time, i.e. divided by 10 time. “E00” is good enough for majority of experiment. When smaller stuff is analyzed, e.g. intracellular vesicle or bacteria, you may need to use “E01”, “E02” or “E03”. When huge cells, e.g. macrophages or dendritic cells, are analyzed, you may need to use “E-1”. In the last column, you will find Mode. When you click the pull down manual, you will see two options, “Lin” and “Log” (not shown here). For P1 and P2, “Lin” is the choice for more than 99% of the experiment; all fluorescent channel, i.e. P3 to P5, P7, “Log” should be the choice. Of course, there is exception. Discuss with the facility staffs when you can not locate your population, may be different scale is needed. D) Putting some plots in the template

1) Before putting the sample into the machine, you may put some plots into the empty template. On the left hand side of the screen, you will see a panel that you may use to put some plots in the template. The most commonly used icon is the “Dot Plot”.

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After you click the “Dot Plot” option, drag an area in the template, an “Inspector” window pops out.

From pull down manual of the “Plot Source”, change it to “Acquisition Analysis”. It means you can acquire the data and analyze the data right after. The other options in this manual allow you to do only one thing. For the first plot, “X Parameter” is always FSC, i.e. Forward Scatter and the “Y Parameter” is always SSC, i.e. Side Scatter. Then click “OK” 2) For the other plots, it depends on how many color(s) you have in your experiment, you can repeat the step (1) above but replacing the “X Parameter” and “Y Parameter” with other colors. In this training, only FL1 (FITC) and FL2 (PE) are used, so one more plot is created.

E) Running sample

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1) You need to set up the voltages (sensitivity of different sensors) and compensation before actual saving your data. Now we go back to the window popped put when you clicked “Connect to Cytometer” under the “Acquire” manual.

When the “Setup” box is checked, nothing is saved. Once you finish setting the voltages and compensation, you need to uncheck this “Setup” box for actually saving the data. 2) In this training, unlabeled beads, FITC beads and PE beads are used. The first sample you put in is unlabeled beads. At first you probably won’t see anything. After playing around the voltages, you would see something as below:

In flow cytometry field, everything is relative, e.g. negative relative to positive. It doesn’t really matter where you put your population. In FSC vs SSC plot above, the population was put in a way that it is under the scale of FSC and SSC. For the FL1 vs FL2, the population was positioned in the lower left corner to leave room for the positive population. 3) Once the voltage is set, click “Pause” (not shown here) in the “Acquisition Control” window, followed by “Abort” (not shown here), uncheck the “Setup” box.

4) Click “Acquire” again, the program is saving the data until the cell (or event) number reaches the number you specified in the “Acquisition & Storage” option under the “Acquire” manual. Once it reaches that number, you will hear the “beep” sound from the computer, meaning the first file is saved. Then in the “Acquisition Control” window, you will see the number of the file change from 001 to 002 implying that the first file is saved and the machine is ready for the second sample.

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5) During the data acquisition, you need to pay attention to the volume left in the sample tube. You can not afford the machine taking up air bubble. When the volume is getting very low and the cell number is not reaching the specified number (for example, if you specified 10,000 events and now only 2,000 is acquired.), you need to click “Pause” in the “Acquisition Control” window to avoid the machine taking air bubble. Remember, once you click “Pause”, you haven’t saved anything yet because you haven’t heard any “beep” sound. Moreover, the file number remains the same. Then you will have 4 choices as shown below:

i) “Resume”: You may add some saline (the core facility has one bottle of saline next to each FACScan) to the sample, put the sample back into the machine and click “Resume”. On top of the 2,000 events, the program would add more to the same file (007 in this case). ii) “Restart”: If you don’t like the acquisition, you may click “Restart”. Then the program would erase 2,000 event acquired previously and restart the acquisition. The file number would remain the same (007 in this case) iii) “Save”: You save this 2,000 events as one file, you will hear the “beep” sound and the file number would increase by 1 (007 to 008) meaning 007 is saved and the machine is ready for the next sample. iv) “Abort”: You erase the 2,000 events and do nothing, the file number remains the same (007 in this case). IMPORTANT: anytime you click “Pause”, think twice before you click any button. Otherwise, you may lose your important data. F) Compensation

1) Now you would put in FITC beads and unlabeled beads. Remember, the FITC beads have only one fluorochrome (color). What you see is shown below:

Unlabeled

FITC

i ii iii iv

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The FITC labeled beads should appear only in the FL1 channel and not in FL2 channel because the beads are labeled with only FITC. This is when the compensation kicks in. The concept of compensation is explained in the slide attached. Simply speaking, you tell the program when signal is detected in FL1, then certain amount of signal needs to be subtracted from FL2. Click the “Compensation” under the “Cytometer” manual.

A “Compensation” window will pop up. In this case, you need to adjust “FL2 - % of FL1”.

But it’s very confusing. Because you have the “FL1- %FL2” and “FL2 - % of FL1”, a way to remember it is: on the right hand side is the single color you put in and on the left hand side is the channel you want to have signal subtraction.

After you adjusted the “FL2 - % of FL1”, you should see the following:

Color youputs in

Signal subtraction

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Now there is no signal in FL2 after subtraction. The compensation introduced above is called “hardware compensation” meaning the machine is really subtracting signal from the raw data. Nowadays, the people in the field use “software compensation” a lot more often than “hardware compensation” because “software compensation” is done after you acquire the raw data. This means you still have the raw data! One commonly used software is “Flowjo” and the core facility is more than happy to give the tutorial about this software if you are interested in. The same principle applies if you have 2, 3 and 4 colors. G) Saving the template and data backup

1) You already saved all the data as you heard all the “beep” sound. You may want to save the template for future use. Go to the “File” followed by “Save as” (not shown here), a new window pops up showing the very same folder you specified in the “Parameter Description”, type in the name you feel it is meaningful. 2) Go to the “Directory” you specified at the very beginning, double check the template file and data file(s). You should see the following under the folder you created:

You will see two different types of icon, one is the template file and the other is data file. 3) You need to transfer the data to storage medium such as USB drive. IMPORTANT MESSAGE: We don’t backup any data and we delete the data once a while, you are the only one responsible for the data. Backup your data right after you are done, don’t expect your data are still here tomorrow.

Unlabeled FITC

Template file Data file

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H) Using the saved “Template” and “Data” from previous experiment

1) Bring your data in the USB drive, click the template icon, you open the “CellQuest” software together with you template. 2) Under “Cytometer” manual, click “Instrument Setting”, a window pops out:

i) Click “Open”, the other window pops out (not shown here). Locate the “Data” file in the USB drive. ii) Then click “Set”, you would see the cursor changed to a “rolling ball”. Once the ball stops rolling, the setting would be changed to that in the “Data” file. iii) Click “Done”, the instrument setting is completed. J) Shutting down the machine

1) Log off your account. You may go to the top left corner and click “log off”, this makes you logging off from your account while leaving the machine on.

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If you are the last user of the day, you need to shut down the machine. Once you click “Shut Down” You would log off from your account and shut down the computer. 2) Clean the machine with 10% bleach (you can see a 50ml tube next to the each Scan area) and water. Fill the tube with red tape with 1ml bleach (never fill more than 1ml, otherwise the sealing part would be eroded), put into the sample dock, flip the fluid control to “Hi” and “Run” for 3 minutes; then flip the arm to either left or right for 10 seconds to clean the waste line. Repeat the same by using water. Then flip the fluid control to “Lo” and “Standby”. Notes: Check the printed schedule, if you are the last person using the machine, shut down both computer and machine.

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DNA cell cycle supplementary notes FACScan training instruction by Flow Cytometry Core Facility of Mount Sinai Last updated 3/9/2010 Notes: i) We assume you completed the basic immunophentyping training on Scan; ii) Always run you sample at low “lo” pressure for the sake of consistence; iii) If propidium iodide (PI) is used as DNA dye, you may use either FL2 or FL3. If 7AAD is used, use FL3; iv) To avoid sample being diluted, when changing sample to sample, don’t switch to standy, leave the machine run. v) This supplement only shows the parts different from the basic training, for step by step procedure, please read carefully the basic training manual. 1) In the “Parameter Description”, do exactly the same as the basic training, except the followings:

i) Each cell (or event), once hits by the laser, generates signal composed of three components: a) area, b) height and c) width (please see slide). For DNA analysis, you want to know the integrated amount of sigal, i.e. area, instead of just height. ii) The width is useful for doublet discrimination. When cells clump together, they take more than to pass through the laser and therefore have wider signal width. The clump makes the event not analyzable and could be gated out by using the width. folder along with other parameters.

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2) In the “Detectors/Amps” window, please pay attention to the followings:

The scale should be changed to “Lin”. Since the G2/M phase only differs from the G0/G1 phase by 2 folds, linear scale is a better scale to resolve the different phases. Check the “DDM” box. Provided that you didn’t turn on the “Rainbow” software, when “DDM” is checked, you can have additional parameter of width and area. Also select either FL2 or FL3 in the “DDM Param” depending on which DNA dye you used. 3) Then go to the “Cytometer” manual and click the “Threshold” option.

A new window pops out, which allows you to specific what parameter you want to use as threshold.

4) You already know how to put the plots in the template area. Put the following plots in the template:

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5) Now you are ready to put your sample into the machine. Most likely you only need to adjust the “FL2 voltage” and the “Threshold” to obtain your pattern.

FSC vs SSC Width vs Area

Histogram: Area

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6) Please be aware that the “Cell quest” software doesn’t have the function to analyze the cell cycle. Training will be provided to use other software for analyzing cell cycle.

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An example of experimental design To determine the percentage, and/or fluorescent intensity of CD3, CD4 and CD8 on FACScan or FACScalibur Tube FL1

FITC FL2 PE

FL3 PerCP

Remarks

1 - - - Unstained for setting the voltage 2 CD3 - - 3 - CD4 - 4 - - CD8

Compensation controls (single stains, one for each fluorochrome used in the experiment)

5 -* CD4 CD8 6 CD3 -* CD8 7 CD3 CD4 -*

Gating Controls (Fluorescence minus one (FMO), leave out one fluorochrome at a time)

8 CD3 CD4 CD8 Experimental control (untreated or healthy sample

for comparison) 9 CD3 CD4 CD8 Experimental sample

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