Flow Cytometry For New PhDs 2012 Simon Monard SCRM and CIR smonard@staffmail.ed.ac.uk Derek Davies CRUK derek.davies@cancer.org.uk Monday 20th and Tues 21st Feb 2012
Program Day 1: Day 2: Basics of flow SM Phenotyping and controls SM Cell Proliferation and death DD Other applications DD Cell Sorting SM New and Parallel Technologies DD Cytometry in Little France SM Experimental Design?? Analysis? Examples Monday 20th and Tues 21st Feb 2012
Basics of Flow Cytometry Simon Monard smonard@staffmail.ed.ac.uk Monday 20th Feb 2012
Flow Cytometers Are instruments that measure the scattered light and fluorescence of particles in suspension that are made to travel in single file in a fluid stream past a light source (laser) either within a quartz flow cell or in a fluid jet in air
Flow Cytometers as biologists we use them largely to distinguish between different types of cells, cells at different stages of cell cycle, death, differentiation, activation etc. The instruments called cell sorters can physically separate cells according those properties.
Particles: Animal and Plant cells, Bacteria, fungi, Pollen, Plankton, Microspheres, Platelets, Organelles Chromosomes Nuclei Etc..
Probes: Tagged antibodies, Fluorescent proteins, DNA dyes, Substrates for enzymes, Microspheres, Reporters of ion and ph Etc
Light
Fluorescence Fluorescence is the phenomena where a material excited with light of one wavelength, reemits light at a longer wavelength 488nm Fluorescein 520nm 400nm 5 0 0 n m 6 0 0 n m
Fluorescent Dyes: Fitc
Fluorescent Dyes: Fitc/PE
Fluorescent Dyes for Labeling Antibodies Fluorescein(Fitc) Phycoerythrin(PE) Allophycocyanin(APC) Peridinin chlorophyll(percp) PE Tandems APC Tandems Nano Crystalls (q-dots) Pacific blue Alexa family E-fluor family Etc etc
Tandem Dyes 488nm PE 585nm 488nm PE CY5 660nm
Nano Crystals: Q-dots, EFnc
Q-Dots
Limitations
Fluorescent Proteins Aequorea victoria
GFP
Dichroic Reflectors DR560LP red green
Optical filters: 585/42BP
Optical filters
Cytometer Components Light Source(s) Method of delivering cells into the light Components to collect light from the cells and deliver to detectors Detectors Components to amplify signals from these detectors. Computer to display data.
Solid State Lasers
Cytometer Components Hydrodynamic focusing Cell suspension PBS PBS
Cytometer Components Interrogation point Laser FSC
Forward Scatter
Interrogation Point
Interrogation Point
Cytometer Components Interrogation point Laser FSC
Cytometer Components Interrogation point Lens
Cytometer Components Interrogation point
Cytometer Components Interrogation point
Side Scatter (90º Scatter)
Side Scatter (90º Scatter)
Light Detection
Schematic of Simple Cytometer FL1 PMT 585/40 520/40 FL2 PMT 560SP Dichroic 488/20 SSC PMT LENS LA SER (4 4 8 8 ) CELL FSC
FACS Calibur 2lasers 4 colour
PE-Cy7 PE-Cy5.5 PE-Cy5 PE-TR PE RFP DsRed mcherry etc
Signal Amplification, Analog Log amp ADC Com puter Display 10000 Preamp FL2-Height 1000 100 10 Lin amp 1 1 10 100 1000 10000 FL1-Height
Signal Amplification, Digital Com puter Display 10000 Preamp DSP FL2-Height 1000 100 10 1 1 10 100 1000 10000 FL1-Height
DATA So each time a cell passes the laser beam, it will generate a signal in all of the detectors this signal goes to an amplifier and then on to an ADC and then to the computer.the computer will accumulate data on a pre-determined number of cells (10,000). The data file generated will have a numerical value for each channel and for each cell.
DATA The data file contains 2 parts, the header and the data FCS2.0 256 1763 1792 372693 0 0 \$BYTEORD\4,3,2,1\$DATATYPE\I\$NEXTDATA\0\$SYS\Macintosh System Software 9.2.2\CREATOR\CELLQuestŖ 3.3\$TOT\26493\$MODE\L\$PAR\7\$P1N\FSC- H\$P1R\1024\$P1B\16\$P1E\0,0\$P2N\SSC-H\$P2R\1024\$P2B\16\$P2E\0,0\$P3N\FL1- H\$P3R\1024\$P3B\16\$P3E\4,0\$P4N\FL2-H\$P4R\1024\$P4B\16\$P4E\4,0\$P5N\FL3- H\$P5R\1024\$P5B\16\$P5E\0,0\$P6N\FL2-A\$P6R\1024\$P6B\16\$P6E\0,0\$P7N\FL2- W\$P7R\1024\$P7B\16\$P7E\0,0\SAMPLE ID\cd6d3+phajkhm\$CYT\FACSCalibur\CYTNUM\E3105\$BTIM\09:09:33\$ETIM\09:10:06\BD$AcqLibVersion\3.1\BD$NPAR\7 \BD$P1N\FSC-H\BD$P2N\SSC-H\BD$P3N\FL1-H\BD$P4N\FL2-H\BD$P5N\FL3-H\BD$P6N\FL2- A\BD$P7N\FL2- W\BD$WORD0\136\BD$WORD1\364\BD$WORD2\537\BD$WORD3\571\BD$WORD4\647\BD$WORD5\405\BD $WORD6\405\BD$WORD7\405\BD$WORD8\405\BD$WORD9\405\BD$WORD10\300\BD$WORD11\322\BD$W ORD12\583\BD$WORD13\0\BD$WORD14\366\BD$WORD15\535\BD$WORD16\586\BD$WORD17\650\BD$WO RD18\100\BD$WORD19\100\BD$WORD20\100\BD$WORD21\100\BD$WORD22\100\BD$WORD23\1\BD$WOR D24\1\BD$WORD25\0\BD$WORD26\0\BD$WORD27\1\BD$WORD28\136\BD$WORD29\52\BD$WORD30\52\B D$WORD31\52\BD$WORD32\52\BD$WORD33\52\BD$WORD34\0\BD$WORD35\213\BD$WORD36\0\BD$WO RD37\0\BD$WORD38\280\BD$WORD39\3\BD$WORD40\3\BD$WORD41\100\BD$WORD42\100\BD$WORD43\ 4\BD$WORD44\1023\BD$WORD45\1023\BD$WORD46\1023\BD$WORD47\54\BD$WORD48\800\BD$WORD49\ 0\BD$WORD50\0\BD$WORD51\52\BD$WORD52\0\BD$WORD53\0\BD$WORD54\0\BD$WORD55\0\BD$WOR D56\0\BD$WORD57\0\BD$WORD58\0\BD$WORD59\0\BD$WORD60\0\BD$WORD61\0\BD$WORD62\0\BD$W ORD63\0\BD$LASERMODE\1\CalibFile\FALSE\P7THRESVOL\52\$FIL\ms1801.003\$DATE\25-Nov-56\
DATA Representation To look at the data from one channel only, it can be presented as a histogram, much like the bar chart here but with many more channels. FREQUENCY (CELL NUMBER) 0 2 4 6 8 10 - +/- + ++ +++ FLUORESCENCE INTENSITY
Histograms CD3 Fitc
To look at two parameters at the same time you can use a pseudo 3-D plot. 3-D Plots
Contour Plots You could use a contour plot. Now you are looking down onto the 3-D plot just like in a geographical map
Dot Plots More popular are dot plots. Each cell is represented by a dot. The frequency information is lost but is implied by the density of dots. F L 2- NORM002 10 4 10 3 10 2 10 1 10 0 10 0 10 1 10 2 10 3 10 4 FL1-H
Gating Strategy And Display 1000 10 4 150 SSC-A 800 600 400 200 74.1 CD4 PE/Cy5 10 3 10 2 10 1 23 # Cells 100 50 83.4 0 0 200 400 600 800 1000 FSC-A 10 0 10 0 10 1 10 2 10 3 10 4 CD3 APC 0 10 0 10 1 10 2 10 3 10 4 CD62L FITC-A
DNA content Flow cytometers are used to measure much more than just fluorescent antibodies. Measuring DNA content gives us information on cell cycle
Calcium Mobilization QuickTime and a Animation decompressor are needed to see this picture.
Calcium Mobilization 2 E6.1 CD6 neg OKT3+MEMpure 86 E6.1 CD6 cyt OKT3+MEMpure 69.3 E6.1 CD6 JP OKT3+MEMpure 51.6 1.5 AlgParm 1 0.5 0 0 50 100 150 200 250 Time: Time (512.00 sec.)
Chromosome Analysis 250 FL1 Chromomycin A3 (CG) 200 150 100 50 0 Hoechst 33342 (AT) 0 50 100 150 200 250 FL2
Chromosome Painting ch15 ch7
How to use them Turn on machine, check fluids. Turn on Computer, open program. Decide where to store your data, file names etc. Make a display, dot plots, histograms. Choose parameters, lin or log. Adjust instrument! Run Samples Clean/shutdown machine.
Find your cells 1000 800 SSC-H: SSC-Height 600 400 Note scale 200 0 0 200 400 600 800 1000 FSC-H: FSC-Height
Gate 1000 800 SSC-H: SSC-Height 600 400 200 0 0 200 400 600 800 1000 FSC-H: FSC-Height
Adjust PMT voltages 10 4 Orange (575/30) 10 3 10 2 10 1 Note scale 10 0 10 0 10 1 10 2 10 3 10 4 Green (525/30)
Summary Flow cytometry is a powerful versatile technology Can be used to demonstrate the difference between biological particles and isolate specific populations Is quite complicated to use right, beware, flow geeks walk among you.