FACSCalibur Training A Look Inside the Box Noor

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

Background of Flow Cytometry SECTION I

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

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

Uses of Flow Cytometry • • It can be used for… • Immunophenotyping • DNA cell cycle/tumor ploidy • Membrane potential • Ion flux • Cell viability • Intracellular protein staining • p. H 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-1980 s

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

Mechanics of a Flow Cytometer (Simplified)

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

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!!

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.

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.

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

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

Laser Beam FSC Detector Original from Purdue University Cytometry Laboratories Collection Lens SSC Detector 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

Granulocytes SSC Lymphocytes Monocytes RBCs, Debris, Dead Cells FSC 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

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.

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

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

Optics- Filters • Different wavelengths of light are scattered simultaneously from a cell Ø 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 Ø Long Pass Ø Short Pass Ø Band Pass Ø Dichroic

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

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

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

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. Detector 1 Dichroic Filter Detector 2

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

Electronics- Photovoltaics • Once the detectors collect photons of light, they convert them to current Ø A voltage pulse is created as cells pass through the laser Ø As the cell passes into the laser, an event window opens Ø More light is scattered as the cell moves into the center of the laser (maxima) Ø As the cell leaves the laser, less and less light is scattered Ø 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

Electronics- The Pulse Photons/Detector (V) Time

Voltage Intensity Pulse Area Pulse Height Pulse Width Time 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

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 Ø 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) Ø Linear amplification is used when there is not such a broad range of signals (e. g. in DNA analysis and calcium flux measurement)

BD FACSCalibur Usage SECTION II

Calibur Platform • The BD FACSCalibur is a modular flow cytometer designed for cell analysis Ø 2 lasers Ø 488 (primary); detects FSC, SSC, and fluorescence Ø 633 (secondary); detects fluorescence Ø Various protocols can be used for cell and DNA analysis Ø Immuno-phenotyping (CD 4, CD 8) Ø Apoptosis (PI and Annexin) Ø Cell Cycle Ø GFP analysis Ø High-throughput drug screening • Flexible system that is very easy to use and is very useful

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

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

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

BD Cell. Quest- Quality Control • Once the instrument has warmed up and ready for use, a QC procedure called “Time Delay Calibration” must be performed Ø Allows the flow cytometer to capture the signals at the right time from each laser as the cell passes through them in succession Ø Should be performed every time a user turns on the machine • Go to File>Open Document browse to Data 1>Setup Folder Ø Choose Time Delay Calibration • Go to Cytometer>Instrument Settings, browse to Data 1>Setup folder Ø Choose IS Time Delay Calibration Ø Choose Set, then Done Ø 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 Ø Choose Time Delay Calibration Ø Select OK when message appears Ø 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. )!!!

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

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

Creating a New Protocol- Plot Types Contour Plot Density Plot Greyscale Density Dot Plot Histogram www. treestar. com

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

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: Ø Rectangular Ø Generally used to define a region of interest; can also be used to define a broad population with outliers Ø Polygonal Ø Used define clustered/dense populations of interest Ø Quadrant Ø 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

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

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

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

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

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

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

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

BD Cell. Quest- Setting Up Controls • After the FSC, SSC, and threshold are adequately set, abort acquisition and uncheck the Setup box Ø Begin acquiring data Ø 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 Ø While running in Setup mode, adjust the parameter voltages to perform compensation

Setting Up Controls- Cell Populations • When adjusted correctly, FSC and SSC should differentiate cell populations ØGranulocytes ØMonocytes ØLymphocytes ØRed Blood Cells/Debris • Based on granularity and size

Setting Up Controls. Compensation • Fluorochromes typically fluoresce over a large part of the spectrum (100 nm 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

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

Setting Up Controls. Compensation • In the Compensation panel, locate the channels requiring compensation (FL 2 -% of FL 1, etc. ) Ø 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 Ø Begin making small adjustments to the percentage Ø After each adjustment, press Stop followed by Restart in either the Parameter Description or Acquisition panel Ø Watch the Mean Fluorescence Intensity (MFI) of the positive control compared to the negative Ø Continue compensating until the mean of the positive population is nearly matched to the mean of the negative ØOnce the means have been approximately matched, abort acquisition, uncheck the Setup box, and begin acquiring data

BD Cell. Quest- Performing Your Experiment • Once all controls are set and compensation has been performed (if necessary), start running unknown samples Ø 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)!!!

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 Ø Keep the cytometer in Run mode Ø 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 Ø 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

Wrap-Up SECTION III

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… Ø Ø Ø Cell. Quest BD FACSDiva Flowjo FCS Express BD Paint-a-Gate • Every program has its Pros and Cons, most require licensing Ø Paint-a-Gate is available on the workstation in the Flow Core

Important Points on Analysis • What kind of data are you looking for? Ø Ø How much fluorescence? What percent are positive? How much more positive is x than y? What is the ratio between param 1 and param 2 • What kind of statistics are available Ø Ø Ø MFI (geometric or arithmetic) %-ages CV Median Anything you can do with a list of numbers

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.

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

Appointments • Flow appointments should be scheduled through one of the Flow Core operators • No appointments by email Ø Call Ben @ x 4277 Ø Stop by HEB 270 or 211 • Please arrive on time Ø Billing begins at the time scheduled Ø 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 24 hrs in advance • Cancellations without proper notice will be billed accordingly unless there are exigent circumstances

References • Numerous References available in the Flow Lab Ø Cytometry Ø Current Protocols in Flow Cytometry Ø Many more reference books available • Purdue University Cytometry Laboratories website: http: //www. cyto. purdue. edu/ Ø Dr. Robert Murphy, Carnegie Mellon University- Basic Theory 1 and 2 Power. Point slides • The Scripps Research Institute Flow Cytometry Core Facility: http: //facs. scripps. edu/

Flow Lab Contact Info… • Noor Khaskhely, M. D. , Ph. D. , Flow Operator Ø noor. khaskely@utoledo. edu • Benjamin Chojnacki, Flow Operator Ø benjamin. chojnacki@utoledo. edu Ø x 4277