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Flow Cytometry & Immunofluorescence(Physics and Chemistry)
Presented by: Diether Recktenwald, PhD
Contact email: [email protected] or [email protected]
Flow Cytometer
From Biomedical Photonics Handbook, Tuan Vo-Dinh ed., CRC Press 2003
Laser delay
Numbers in Memory
FSC
SSC
FITC
APC
309
313
337
336
367
364
393
316
315
399
450
1049
2998
7243
1272
015
405
1265
272
4830
2010
3746
436
143
634
930
933
933
040
444
941
933
340
330
733
833
041
843
941
542
530
332
540
643
136
240
739
040
7
445
378
372
373
450
441
459
399
456
319
445
1009
3089
7640
1353
416
372
1357
076
1831
2610
0443
337
334
944
335
139
339
242
645
544
137
532
939
742
739
242
936
130
338
036
131
443
931
341
636
734
635
5
351
383
375
406
377
318
367
319
375
423
432
937
2397
5786
1019
512
300
1025
657
9124
7184
243
333
131
130
837
634
941
432
337
330
331
433
841
830
731
735
331
330
340
337
830
840
640
530
335
340
532
8
403
340
387
360
353
401
387
353
410
389
402
393
356
312
389
391
410
502
915
2372
5860
1015
112
370
1022
058
3124
7886
139
439
137
640
831
433
741
936
838
540
437
533
235
739
336
435
431
337
439
539
8
Flow Cytometry Data Analysis
Cell P1 P2 P3 P4 P5 Pop#1 242 135 704 175 612 12 146 132 690 178 566 13 269 147 89 206 580 34 442 143 399 250 255 45 212 167 155 926 526 26 269 2 659 207 575 17 204 232 112 171 679 38 152 74 160 828 532 2
...9997 215 119 138 936 662 29998 244 50 72 261 543 39999 214 137 174 1014 597 2
10000 312 87 110 904 560 2
30020010000
10
20
30
Event histogram
P3 intensity
# ev
ents
30020010000
100
200
300
"Dotplot"
P3
P4
Note: more than 12 parameters in advanced FACS systems
“Gating”
From Biomedical Photonics Handbook, Tuan Vo-Dinh ed., CRC Press 2003
Dead
Live
Flow Sorting and Plate Detection
Cell Sorting for Functional Studies
Parameters For Cell Analysis by Flow Cytometry
Analyse and Sort based on:• light scatter• immunofluorescence• fluorescent in-situ hybridization• DNA content• transfection with fluorescent proteins• protein content• auto-fluorescence• enzyme activity• pH• redox potential• other components detectable by fluorescence Hela cells transfected with fluorescent protein
vectors for nuclei, mitochondria and tubulin.
Immunofluorescence
• Sample conditioning• Disaggregation of tissues• Pre-enrichment
• Reaction of sample with reagent• Direct or indirect
immunofluorescence• Wash or no-wash
• Multi-color fluorescence measurement
• Data analysis
Immunofluorescence Data (1)
Immunofluorescence (1) Limit of Detection for Rare Cells
Gross HJ et al, Cytometry. 1993;14(5):519-26
10-6 10-5 control
Routine >0.2%
Optimized instrument >0.01%
Optimized system >10-7
Immunofluorescence Data (2)
Immunofluorescence Data (3)
140014000 37000
180000PEQuantitation
Molecule #/cell
CD3 8.1 x 104 CD4 5.9 x 104 CD8 1.4 x 105
CD11a 2.7 x 104 CD16 7.9 x 104 CD18 3.1 x 104 CD45 1.9 x 105
From: Appendix A, Cell Separation Methods and Applications. Marcel Dekker 1998. Recktenwald D and Radbruch A, eds.
Sensitivity
Microsphere-based assays for soluble analytes
IgG 2b 20 ng
IgG 1 Bead
IgG 2a Bead
IgG 2b Bead
IgG 3 Bead
IgM Bead
IgE Bead
IgA Bead
IgE 20 ng
IgG1 Bead
IgG2a Bead
IgG2b Bead
IgG3 Bead
IgM Bead
IgE Bead
IgA Bead
Dr. Rudi Varro, BD Biosciences
Immunofluorescence Issues
• Label selection (sensitivity and compensation)
• Photobleaching (especially energy transfer conjugates)
• Environment sensitive fluorescence (i.e.FITC)
• Fixation• Dead cells (PI, EMA)
• Reagent equilibrium binding• Binding kinetics• “Non-specific” reactions
Ligand binding (1)
Ligand binding (2)
Effect of "non-specific" binding
0
0.2
0.4
0.6
0.8
1
0 10 20 30 40 50
Concentration
Inte
nsity
ImmunofluorescenceMulti-color Analysis
From Biomedical Photonics Handbook, Tuan Vo-Dinh ed., CRC Press 2003
Six color example
• CD3 FITC• CD56 PE• CD8 PE-Texas Red• CD19 PE-Cy7• CD14 APC• CD4 APC-Cy7
End
address questions to:Diether Recktenwald PhD
BD Biosciences Immunocytometry Systems
2350 Qume Dr.
San Jose CA 95131-1807, USA
Phone: 408-954-2191(o)
FAX: 928-441-2245(efax)
Email: [email protected] or
