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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 Slide 2 Background of Flow Cytometry SECTION I Slide 3 Experimental design Sample preparation Choosing the proper instrument Setting up the instrument Collecting the proper data Interpreting the data Graphics presentation and publication Sorting The Many Parts of Flow Specific Applications Courses Flow Basics Data Analysis Slide 4 Cytometry v. Flow Cytometry Cytometry Localization of antigen is possible Poor enumeration of cell subtypes Limiting number of simultaneous measurements Flow Cytometry No localization of antigens Enumeration of subtypes via cell/size/granularity/ markers Can look at numerous parameters simultaneously at high rate Slide 5 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 Uses of Flow Cytometry Slide 6 Optics Fluidics Electronics 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 Mechanics of a Flow Cytometer Slide 7 Mechanics of a Flow Cytometer (Simplified) Slide 8 Fluidics- Simplified Schematic Sheath Tank Waste Tank Line Pressure Vacuum Sample Pressure (Variable) Sheath Pressure (Constant) Sample Tube Slide 9 10 psi 10.2 psi 10 psi 10.4 psi 10 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 CVs and increase in multiple cells passing through the laser at once Faster cell analysis BUT loss of resolution!! Fluidics- Sample Differential Slide 10 Effect of Flow Rate on Sample Data Resolution With increased sample pressure, flow rate increases resulting in a decrease in data resolution. Low pressure High pressure Slide 11 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- Light Scatter Slide 12 FSC Detector Laser Beam Original from Purdue University Cytometry Laboratories Optics- Forward Scatter Slide 13 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 Interrogation- Forward Scatter Slide 14 Original from Purdue University Cytometry Laboratories FSC Detector Collection Lens SSC Detector Laser Beam 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 Optics- Side Scatter Slide 15 Since FSC size and SSC internal structure, a correlated measurement between them can allow for differentiation of cell types in a heterogeneous cell population FSC SSC Lymphocytes Monocytes Granulocytes RBCs, Debris, Dead Cells Why Look at FSC v. SSC Slide 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. Slide 17 Optics- Detectors There are two main types of photo detectors used in flow cytometry Photodiodes o 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. Slide 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 Slide 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 Slide 20 Transmit all wavelengths greater than specified wavelength Example: 500LP will transmit all wavelengths greater than 500nm Optics- Long Pass Filters 400nm 500nm 600nm 700nm Transmittance Original from Cytomation Training Manual Slide 21 Transmits all wavelengths less than specified wavelength Example: 600SP will transmit all wavelengths less than 600nm. Optics- Short Pass Filter 400nm 500nm 600nm 700nm Transmittance Original from Cytomation Training Manual Slide 22 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) Optics- Band Pass Filter 400nm 500nm 600nm 700nm Transmittance Original from Cytomation Training Manual Slide 23 Long pass or short pass filters Placed at a 45 angle of incidence Part of the light reflected at 90, and part of the light is transmitted and continues on. Optics- Dichroic Filters Dichroic Filter Detector 1 Detector 2 Slide 24 Spectra of Common Fluorochromes Common fluorochromes used for violet, blue, and red laser flow cytometry From BD Multicolor Fluorochrome Reference Chart Slide 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 Slide 26 Time Photons/Detector (V) Electronics- The Pulse Slide 27 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 Slide 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) Slide 29 SECTION II BD FACSCalibur Usage Slide 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 Slide 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 Slide 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 Slide 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 Slide 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 Choose Time Delay Calibration 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.)!!! Slide 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 Slide 36 Creating a New Protocol- Plot Types There are several plot choices depending upon the experiments 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 Slide 37 Creating a New Protocol- Plot Types Contour Plot Density Plot Greyscale Density Dot Plot www.treestar.com Histogram Slide 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 Slide 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 Slide 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 investigators folder Give the protocol an appropriate name Slide 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>(PIs 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 Slide 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.)!!! Slide 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 Slide 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 2 nd decade Slide 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 Slide 46 FSC Detector FSC Detector Time Threshold (eg. 52) Threshold (eg. 52) Setting Up Controls- Threshold Slide 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 Slide 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 Slide 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 Slide 50 Setting Up Controls- Compensation 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+ Control FITC Overlapping into the PE Channel FITC Properly Compensated Slide 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 Slide 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)!!! Slide 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 Slide 54 SECTION III Wrap-Up Slide 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 Slide 56 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 Important Points on Analysis Slide 57 Everythings Relative The relative bin numbers are just thatrelative. 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. Slide 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: DONT 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 Slide 59 Appointments Flow appointments should be scheduled through one of the Flow Core operators No appointments by email Call Ben @ 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 Slide 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: http://www.cyto.purdue.edu/ http://www.cyto.purdue.edu/ Dr. Robert Murphy, Carnegie Mellon University- Basic Theory 1 and 2 PowerPoint slides The Scripps Research Institute Flow Cytometry Core Facility: http://facs.scripps.edu/ http://facs.scripps.edu/ Slide 61 Flow Lab Contact Info Noor Khaskhely, M.D., Ph.D., Flow Operator noor.khaskely@utoledo.edu Benjamin Chojnacki, Flow Operator benjamin.chojnacki@utoledo.edu x4277