Flow Cytometry For New PhDs

Transcription

1 Flow Cytometry For New PhDs 2012 Simon Monard SCRM and CIR Derek Davies CRUK Monday 20th and Tues 21st Feb 2012

2 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

3 Basics of Flow Cytometry Simon Monard Monday 20th Feb 2012

4 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

5 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.

6 Particles: Animal and Plant cells, Bacteria, fungi, Pollen, Plankton, Microspheres, Platelets, Organelles Chromosomes Nuclei Etc..

7 Probes: Tagged antibodies, Fluorescent proteins, DNA dyes, Substrates for enzymes, Microspheres, Reporters of ion and ph Etc

8 Light

9 Fluorescence Fluorescence is the phenomena where a material excited with light of one wavelength, reemits light at a longer wavelength 488nm Fluorescein 520nm 400nm n m n m

10 Fluorescent Dyes: Fitc

11 Fluorescent Dyes: Fitc/PE

12 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

13 Tandem Dyes 488nm PE 585nm 488nm PE CY5 660nm

14 Nano Crystals: Q-dots, EFnc

15 Q-Dots

16 Limitations

17 Fluorescent Proteins Aequorea victoria

18 GFP

19

20 Dichroic Reflectors DR560LP red green

21 Optical filters: 585/42BP

22 Optical filters

23 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.

24

25 Solid State Lasers

26 Cytometer Components Hydrodynamic focusing Cell suspension PBS PBS

27 Cytometer Components Interrogation point Laser FSC

28 Forward Scatter

29 Interrogation Point

30 Interrogation Point

31 Cytometer Components Interrogation point Laser FSC

32 Cytometer Components Interrogation point Lens

33 Cytometer Components Interrogation point

34 Cytometer Components Interrogation point

35 Side Scatter (90º Scatter)

36 Side Scatter (90º Scatter)

37 Light Detection

38 Schematic of Simple Cytometer FL1 PMT 585/40 520/40 FL2 PMT 560SP Dichroic 488/20 SSC PMT LENS LA SER ( ) CELL FSC

39 FACS Calibur 2lasers 4 colour

40 PE-Cy7 PE-Cy5.5 PE-Cy5 PE-TR PE RFP DsRed mcherry etc

41 Signal Amplification, Analog Log amp ADC Com puter Display Preamp FL2-Height Lin amp FL1-Height

42 Signal Amplification, Digital Com puter Display Preamp DSP FL2-Height FL1-Height

43 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.

44 DATA The data file contains 2 parts, the header and the data FCS \$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\ms \$DATE\25-Nov-56\

45 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) / FLUORESCENCE INTENSITY

46 Histograms CD3 Fitc

47 To look at two parameters at the same time you can use a pseudo 3-D plot. 3-D Plots

48 Contour Plots You could use a contour plot. Now you are looking down onto the 3-D plot just like in a geographical map

49 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- NORM FL1-H

50 Gating Strategy And Display SSC-A CD4 PE/Cy # Cells FSC-A CD3 APC CD62L FITC-A

51 DNA content Flow cytometers are used to measure much more than just fluorescent antibodies. Measuring DNA content gives us information on cell cycle

52 Calcium Mobilization QuickTime and a Animation decompressor are needed to see this picture.

53 Calcium Mobilization 2 E6.1 CD6 neg OKT3+MEMpure 86 E6.1 CD6 cyt OKT3+MEMpure 69.3 E6.1 CD6 JP OKT3+MEMpure AlgParm Time: Time ( sec.)

54 Chromosome Analysis 250 FL1 Chromomycin A3 (CG) Hoechst (AT) FL2

55 Chromosome Painting ch15 ch7

56 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.

57 Find your cells SSC-H: SSC-Height Note scale FSC-H: FSC-Height

58 Gate SSC-H: SSC-Height FSC-H: FSC-Height

59 Adjust PMT voltages 10 4 Orange (575/30) Note scale Green (525/30)

60 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.