1. FACSCalibur Training A Look Inside the Box
Noor Khaskhely, M.D., Ph.D. (Operator)
Benjamin Chojnacki (Operator) Flow Cytometry Core Lab
University of Toledo College of Medicine

2. Background of Flow Cytometry
Section I

3. The Many Parts of Flow Experimental design Sample preparation
Choosing the proper instrument
Setting up the instrument
Collecting the proper data
Interpreting the data
Graphics presentation and publication
Sorting
Flow Basics
Data Analysis
Specific Applications Courses

4. Cytometry v. Flow Cytometry
Localization of antigen is possible
Poor enumeration of cell subtypes
Limiting number of simultaneous measurements
No localization of antigens
Enumeration of subtypes via cell/size/granularity/ markers
Can look at numerous parameters simultaneously at high rate

5. Uses of Flow Cytometry It can be used for…
Immunophenotyping
DNA cell cycle/tumor ploidy
Membrane potential
Ion flux
Cell viability
Intracellular protein staining
pH changes
Cell tracking and proliferation
Sorting
Redox state
Chromatin structure
Total protein
Lipids
Surface charge
Membrane fusion/runover
Enzyme activity
Oxidative metabolism
Sulfhydryl groups/glutathione
DNA synthesis
DNA degradation
Gene expression
The use of flow in research has boomed since the mid-1980s

6. Mechanics of a Flow Cytometer
Cells in suspension are brought in single file past
a focused laser where they scatter light and emit fluorescence that is filtered and collected
then converted to digitized values that are stored in a file for analysis
Fluidics
Optics
Electronics

7. Mechanics of a Flow Cytometer (Simplified)

8. Fluidics- Simplified Schematic
Sheath
Tank
Waste
Line Pressure
Vacuum
Sample
Pressure
(Variable)
Sheath Pressure
(Constant)
Tube

9. Fluidics- Sample Differential
10 psi
10.2 psi
10.4 psi
10.8 psi
Difference in pressure between sample and sheath
This will control sample volume flow rate
The greater the differential, the wider the sample core.
If differential is too large, cells will no longer line up single file
Results in wider CV’s and increase in multiple cells passing through the laser at once » » » Faster cell analysis BUT loss of resolution!!

10. Effect of Flow Rate on Sample Data Resolution
Low pressure
High pressure
Effect of Flow Rate on Sample Data Resolution
With increased sample pressure, flow rate increases resulting in a decrease in data resolution.

11. Optics- Light Scatter
When light from a laser interrogates a cell, that cell scatters light in all directions.
The scattered light can travel from the interrogation point down a path to a detector.

12. Optics- Forward Scatter
FSC Detector
Laser Beam
Original from Purdue University Cytometry Laboratories

13. Interrogation- Forward Scatter
Light that is scattered in the forward direction (along the same axis the laser is traveling) is detected in the Forward Scatter Channel.
The intensity of this signal has been attributed to cell size, refractive index (membrane permeability)
Forward Scatter=FSC=FALS=LALS

14. FSC Detector
Collection
Lens
SSC
Detector
Laser Beam
Original from Purdue University Cytometry Laboratories
Optics- Side Scatter
Laser light that is scattered at 90 degrees to the axis of the laser path is detected in the Side Scatter Channel. The intensity of this signal is proportional to the amount of cytosolic structure in the cell (eg. granules, cell inclusions, etc.)– Side Scatter=SSC=RALS=90° Scatter

15. Why Look at FSC v. SSC Granulocytes Lymphocytes Monocytes
RBCs, Debris,
Dead Cells
Why Look at FSC v. SSC
Since FSC ≈ size and SSC ≈ internal structure, a correlated measurement between them can allow for differentiation of cell types in a heterogeneous cell population

16. Optics- Fluorescence Channels
As the laser interrogates the cell, fluorochromes on/in the cell (extrinsic or intrinsic) may absorb some of the light and become excited
Fluorochromes leave their excited state and release energy in the form of a photon with a specific wavelength, longer than the excitation wavelength
Emitted photons pass through the collection lens and are split and steered down specific channels with the use of filters.

17. Optics- Detectors
There are two main types of photo detectors used in flow cytometry
Photodiodes
Used for strong signals, when saturation is a potential problem (eg. FSC detector)
Photomultiplier tubes (PMT)
More sensitive than a Photodiode, a PMT is used for detecting small amounts of fluorescence emitted from fluorochromes.

18. Optics- Fluorescence Detectors
FSC Detector
Collection
Lens
Laser Beam
Fluorescence
Detector A, B, C, etc…
Original from Purdue University Cytometry Laboratories, Modified by Benjamin Chojnacki

19. Optics- Filters
Different wavelengths of light are scattered simultaneously from a cell
Need to split the light into its specific wavelengths in order to measure and quantify them independently. This is done with filters
Optical filters are designed such that they absorb or reflect some wavelengths of light, while transmitting others
3 types of filters
Long Pass
Short Pass
Band Pass
Dichroic

20. Optics- Long Pass Filters
Transmit all wavelengths greater than specified wavelength
Example: 500LP will transmit all wavelengths greater than 500nm
400nm
500nm
600nm
700nm
Transmittance
Original from Cytomation Training Manual

21. Optics- Short Pass Filter
Transmits all wavelengths less than specified wavelength
Example: 600SP will transmit all wavelengths less than 600nm.
400nm
500nm
600nm
700nm
Transmittance
Original from Cytomation Training Manual

22. Optics- Band Pass Filter
Transmits a specific band of wavelengths
Example: 550/20BP Filter will transmit wavelengths of light between 540nm and 560nm (550/20 = 550+/-10, not 550+/-20)
400nm
500nm
600nm
700nm
Transmittance
Original from Cytomation Training Manual

23. Optics- Dichroic Filters
Long pass or short pass filters
Placed at a 45º angle of incidence
Part of the light reflected at 90º , and part of the light is transmitted and continues on.
Dichroic Filter
Detector 1
Detector 2 .

24. Spectra of Common Fluorochromes
Common fluorochromes used for violet, blue, and red laser flow cytometry
From BD Multicolor Fluorochrome Reference Chart

25. Electronics- Photovoltaics
Once the detectors collect photons of light, they convert them to current
A voltage pulse is created as cells pass through the laser
As the cell passes into the laser, an event window opens
More light is scattered as the cell moves into the center of the laser (maxima)
As the cell leaves the laser, less and less light is scattered
After a set amount of time, the window closes until another object enters the beam
The current is processed into signal that is then converted into a digitized value

26. Electronics- The Pulse
Time
 Photons/Detector (V)

27. Digitization of the Pulse
Pulse Height
Pulse Width
Pulse Area
Time
Voltage Intensity
Digitization of the Pulse
Each pulse has a height (maximum voltage) and area. Area scaling can be performed to assure that data is linear, allowing comparisons to be made between data sets

28. Electronics- Linear and Log Amplifiers
When the current exits the detector, it passes through either a linear or log amplifier where it is converting it into the voltage pulse.
Intensity of the voltage can be adjusted by amplifying it on a linear scale or converting it to a logarithmic scale
The use of a log amp is beneficial when there is a broad range of fluorescence as that may need to be compressed (this is generally true of most biological distributions)
Linear amplification is used when there is not such a broad range of signals (e.g. in DNA analysis and calcium flux measurement)

29. BD FACSCalibur Usage
Section II

30. Calibur Platform
The BD FACSCalibur is a modular flow cytometer designed for cell analysis
2 lasers
488 (primary); detects FSC, SSC, and fluorescence
633 (secondary); detects fluorescence
Various protocols can be used for cell and DNA analysis
Immuno-phenotyping (CD4, CD8)
Apoptosis (PI and Annexin)
Cell Cycle
GFP analysis
High-throughput drug screening
Flexible system that is very easy to use and is very useful

31. Calibur Platform- Fluorescence Detectors
488 Laser (Blue) has 3 parameters/filters to choose from
FL-1; 530/30
FL-2; 585/42
FL-4; 661/16
635 Laser (Red) has 1 parameter/filter
FL-3; 670LP

32. FACSCalibur- Start-Up
Fluidics should be started first
Make sure there is an appropriate amount of sheath fluid and that the waste container is not full
Alarms will sound if there is a problem with either container during startup
Press the green button in the middle-right of the fluidics cart beneath the FACSCalibur bench to start pump
Flow cytometer should be started second
Press the green button on the right side of the Calibur to turn on the machine
The computer should always be started last
If the computer is already on, be sure to restart
Login: Administrator
Password: BDIS
Always allow the instrument 15 minutes to warm-up prior to use

33. BD CellQuest Pro
Once logon is complete, find the menu icons displayed at the bottom-center and open BD CellQuest Pro
A new blank document will open
Go to Acquire> Connect to Cytometer
Connects the instrument to the computer
Go to Cytometer> Detectors and Amps, then return to the Cytometer menu and choose Compensation, Threshold, and Status
These main palettes allow you to control the instrument
The status palette will display if the instrument is ready for use

34. BD CellQuest- Quality Control
Once the instrument has warmed up and ready for use, a QC procedure called “Time Delay Calibration” must be performed
Allows the flow cytometer to capture the signals at the right time from each laser as the cell passes through them in succession
Should be performed every time a user turns on the machine
Go to File>Open Document browse to Data 1>Setup Folder
Choose Time Delay Calibration
Go to Cytometer>Instrument Settings, browse to Data 1>Setup folder
Choose IS Time Delay Calibration
Choose Set, then Done
This will load instrument settings for the calibration
Load calibration beads onto the bulk injection port and press Run on the front of the cytometer
Click Acquire, then go to Cytometer
Select OK when message appears
A sound will acknowledge that the operation was performed successfully
Unload the calibration beads
Press the Standby button on the front of the cytometer
The cytometer is now ready for use (HUZZAH, etc.)!!!

35. BD CellQuest- Creating a New Protocol
If using a premade template, close the window that opened when CellQuest launched
If the window was closed inadvertently, go to File>New Document
In order to acquire data, plots need to be created
Go to Plot and choose the appropriate type
Go to Windows >Inspector
This controls the formatting of the plots

36. Creating a New Protocol- Plot Types
There are several plot choices depending upon the experiment’s application and data being captured
Single Color Histogram
Fluorescence intensity (FI) versus count
Two Color Dot Plot
FI of parameter 1 versus FI of Parameter 2
Two Color Contour Plot
FI of P1 versus FI of P2. Concentric rings form around populations. The more dense the population, the closer the rings are to each other
Two Color Density Plot
FI of P1 versus FI of P2. Areas of higher density will have a different color than other areas

37. Creating a New Protocol- Plot Types
Contour Plot
Density Plot
Greyscale Density
Dot Plot
Histogram

38. BD CellQuest- Creating a New Protocol
Once a plot type is selected, it will need to be formatted properly
With the plot highlighted choose Acquisition to Analysis from Plot Type in the Inspector
Choose the appropriate parameters (or parameter for histogram plots) for the axes
Repeat until there are an appropriate number of plots for the analysis
This will be based on the needs of each project
Place gates in each plot to distinguish areas of interest
Since there is no data, gates can be placed in the plot where data is expected to be
Gates can be adjusted during data acquisition

39. Creating a New Protocol- Gating
Gates are used to isolate subsets of cells or “populations” on a plot
Allows the ability to look at parameters specific to only that subset
Can use boolean logic to include or exclude multiple gates
For scatter plots there are 3 main types of gates:
Rectangular
Generally used to define a region of interest; can also be used to define a broad population with outliers
Polygonal
Used define clustered/dense populations of interest
Quadrant
Most often used in multicolor to distinguish negative populations from single- and double-positive ones
For histograms bar/horizontal gates are used to define spikes/peaks of interest

40. BD CellQuest- Creating a New Protocol
Once gates have been set around areas of interest, stats can be displayed
Highlight a plot and choosing Histogram or Quadrant (depends on the plot type)
Stat boxes can be edited to display any number of items pertinent to the experiment/project
Statistics can also be displayed for a plot without gates if needed
Once the plots are in place, go to File>Save Document As
Navigate to the appropriate investigator’s folder
Give the protocol an appropriate name

41. Creating a New Protocol- Naming Folders
When performing analysis a new folder should be made to contain the data from that run
Go to Acquire>Parameter Description
Palette allows control of instrument, naming of folders and sample (Sample ID)
On the Acquisition Palette go to the Directory line and choose Change
In the navigation window go to Data 1>Sample Files C1>(PI’s Folder)
Choose New Folder
The new folder should be named to include:
Date (including year)
Your Initials
A new folder should be made for each day of use

42. Creating a New Protocol- Naming Files
The default name for each file can be changed if more specification is needed
On the Acquisition Palette go to File line and choose Change
In the Custom Prefix line specify the name the files should have
In the File line enter the desired suffix number of the first file
This number will be incremented with each sample as data is saved
Everything is prepared for acquiring data (HUZZAH, etc.)!!!

43. BD CellQuest- Controls
Every experiment performed on the cytometer should include controls
Just plain, good ol’ fashioned science
Distinguish cell populations from debris or background
Allow differentiation of labeled cells (i.e., cell subtypes)
Necessary controls will depend on the experiment
Common controls include
Single negative controls
Unlabeled cells
Isotype controls
Fluorescence minus one (FMO)
Single positive controls
One for each color being used
BD Comp Beads

44. BD CellQuest- Setting Up Controls
Start acquiring data with Negative or Isotype controls
In the Acquisition palette check the Setup box next to the acquisition controls
Place the control tube on the bulk injection port
Press the Run button on the front of the cytometer and click Acquire
Adjust the voltage of the appropriate parameters
If there is debris, adjust the threshold
Data should placed so that it is easily viewable without going beyond the 2nd decade

45. Setting Up Controls- Threshold
When the laser interrogates an object, light is scattered.
If the amount of light scattered surpasses a threshold, then the electronics opens a set window of time for signal detection
The threshold can be set on any parameter, but is usually set on FSC
This is generally used to eliminate debris or cell fragments that are not of interest

46. Setting Up Controls- Threshold
FSC
Detector
Time
Threshold
(eg. 52)

47. BD CellQuest- Setting Up Controls
After the FSC, SSC, and threshold are adequately set, abort acquisition and uncheck the Setup box
Begin acquiring data
Data will be automatically saved based on the settings performed
Once negative controls have been recorded, repeat the process with single positive controls or FMOs
While running in Setup mode, adjust the parameter voltages to perform compensation

48. Setting Up Controls- Cell Populations
When adjusted correctly, FSC and SSC should differentiate cell populations
Granulocytes
Monocytes
Lymphocytes
Red Blood Cells/Debris
Based on granularity and size

49. Setting Up Controls- Compensation
Fluorochromes typically fluoresce over a large part of the spectrum (100nm or more)
Depending on filter arrangement, a detector may see some fluorescence from more than 1 fluorochrome. (referred to as bleed over)
Compensation needs to be made for this bleed over so that 1 detector reports signal from only 1 fluorochrome

50. Setting Up Controls- Compensation
FITC+ Control
Parameter voltage must be altered to compensate for this overlap
Proper compensation used to be performed visually, but this has been found to be very wrong
In order to properly ensure compensation, must use Mean Matching
FITC Overlapping into the PE Channel
FITC Properly Compensated

51. Setting Up Controls- Compensation
In the Compensation panel, locate the channels requiring compensation (FL2-% of FL1, etc.)
The positive control you are using is the first channel being subtracted from the second (FITC from PE as above)
Place the positive control tube onto the bulk injection port check the Setup box and click Acquire
Begin making small adjustments to the percentage
After each adjustment, press Stop followed by Restart in either the Parameter Description or Acquisition panel
Watch the Mean Fluorescence Intensity (MFI) of the positive control compared to the negative
Continue compensating until the mean of the positive population is nearly matched to the mean of the negative
Once the means have been approximately matched, abort acquisition, uncheck the Setup box, and begin acquiring data

52. BD CellQuest- Performing Your Experiment
Once all controls are set and compensation has been performed (if necessary), start running unknown samples
Be sure that the Setup box is left unchecked
Continue acquiring until all samples have been run
If another appointment follows yours, do not shutdown the cytometer
Place a tube of water on the sample injection port and allow it to run for ~2 minutes.
Press the Standby button on the front of the cytometer
You may now skip away in bliss at having successfully revolutionized your field of research with your staggeringly awesome data (DOUBLE HUZZAH)!!!

53. FACSCalibur- Shut Down
If you are the last appointment of the day, once all samples are run, place a tube of sterile water onto the sample injection port
Keep the cytometer in Run mode
Leave the tube on for ~2 minutes
Replace the tube of water with the tube containing 10% bleach for ~2 minutes
Place the tube containing water back onto the cytometer for another 2 minutes
Place the cytometer in Standby mode
Turn the computer off
Go to File>Shutdown
Turn the instrument off by pressing the illuminated green button on the back, right side of the cytometer
Switch off the fluidics cart by pressing the illuminated green button

54. Wrap-Up
Section III

55. Interpretation
Once the values for each parameter are in a list mode file, specialized software can graphically represent it.
The data can be displayed in 1, 2, or 3 dimensional format
Common programs include…
CellQuest
BD FACSDiva
Flowjo
FCS Express
BD Paint-a-Gate
Every program has its Pros and Cons, most require licensing
Paint-a-Gate is available on the workstation in the Flow Core

56. Important Points on Analysis
What kind of data are you looking for?
How much fluorescence?
What percent are positive?
How much more positive is x than y?
What is the ratio between param1 and param2
What kind of statistics are available
MFI (geometric or arithmetic)
%-ages CV
Median
Anything you can do with a list of numbers

57. Everything’s Relative
The relative bin numbers are just that…relative.
Saying your cells have a mean fluorescence intensity of 100 means absolutely nothing until you compare it to a negative.
The fact that everything is relative allows you to compare 2, 3, or 20 samples using the same instrument settings.

58. FACSCalibur- Troubleshooting
Under normal weekday circumstances, the cytometer should be ready for use
Contact one of the operators to change the sheath or empty the waste
If using the instrument on the weekend, set up will need to be performed
If the sheath is empty, Refill the sheath from one of the BD FACSFlow containers designated
If the waste is full , empty the container into the sink and reconnect the cap/tubing
If any issues with the cytometer are encountered:
DON’T PANIC!! (Always carry a towel)
Remove any sample running
Place the cytometer in Standby
Contact one of the operators
Do not try to fix the instrument under any circumstance
We have a service contract for that 

59. Appointments
Flow appointments should be scheduled through one of the Flow Core operators
No appointments by
Call x4277
Stop by HEB 270 or 211
Please arrive on time
Billing begins at the time scheduled
Additional time for use will be allowed only if no other users are scheduled following the appointment
Billing is done in 15 minute increments
Cancellations should be made at least 24hrs in advance
Cancellations without proper notice will be billed accordingly unless there are exigent circumstances

60. References Numerous References available in the Flow Lab
Cytometry
Current Protocols in Flow Cytometry
Many more reference books available
Purdue University Cytometry Laboratories website:
Dr. Robert Murphy, Carnegie Mellon University- Basic Theory 1 and 2 PowerPoint slides
The Scripps Research Institute Flow Cytometry Core Facility:

61. Flow Lab Contact Info…
Noor Khaskhely, M.D., Ph.D., Flow Operator
Benjamin Chojnacki, Flow Operator
x4277