Applications Flow Cytometry

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1 Applications Flow Cytometry Genetically encoded fluorescent reporter proteins (transfection-, infection-, induction- rates, expression, deletion ) GFP Immunostainings of cell-specific antigens (extra- and intracellular) DNA stainings (cell cycle, ploidy, apoptosis/necrosis) Functional assays and Kinetics (apoptosis, cell proliferation, cell signaling: Ca 2+ -flux) Used by Biooptics with permission of Tanja Schwickert

2 Genetically encoded fluorescent proteins report for Transfection Infection GFP Gene dsred Expression Induction Deletion 23% Protein interaction (FRET, split-gfp) GFP Location (Photoactivatable GFP) +Dox dsred GFP Used by Biooptics with permission of Tanja Schwickert

3 Genetically encoded fluorescent proteins report for Transfection Infection Split-GFP Protein A B Expression Induction Deletion NGFP CGFP Protein interaction (FRET, split-gfp) Location (Photoactivatable GFP) GFP Used by Biooptics with permission of Tanja Schwickert

4 Genetically encoded fluorescent proteins report for Transfection Infection Expression Induction Deletion Protein interaction (FRET, split-gfp) Location (Photoactivatable GFP) 405nm (1P) 830nm (2P) + CO 2 488nm Victora et al 2011

5 Immunophenotyping by cell-specific surface staining SSC-A Live cells FSC-A SSC-H Singlets SSC-W Empty ch -Auto -fluorescence Fluorescent ch Bone Marrow

6 Immunophenotyping by cell-specific surface staining Live, Singlets CD11b-PeCy7 CD11c-BV421 Ter119-BV510 Gr1-APC Ly6c-PE Ly6c-Pe C-Kit-PerCP-Cy5.5 B220-Fitc B220-Fitc Used by Biooptics with permission of Tanja Schwickert

7 Immunophenotyping: Multi-color FACS Laser 1: 405nm Laser2: 488nm Laser3: 561nm Laser4: 640nm X-color 450/50 PacBlue BV /50 BV /40 BV /60 BV /30 Fitc AF488 GFP 695/40 PerCP- Cy /15 PE 610/20 PE- CF /60 PE-Cy7 670/30 APC A647 4 x x x x 5 x x x x x 6 x x x x x x 7 x x x x x x x 8 x x x x x x x x 9 x x x x x x x x x 10 x x x x x x x x x x 11 x x x x x x x x x x x Used by Biooptics with permission of Tanja Schwickert 780/60 APC-Cy7 Laser & filter settings: Fortessa

8 Phenotyping and functionality by cytoplasmic-, nuclear staining Staining of surface antigen Fixation Permeabilization Y Y Intracellular stainings Cytoplasmic Igκ (cytoplasmic) Staining of cytoplasmic & nuclear antigens Y Y Nuclear Aiolos (transcription factor) B220-BV421 (surface) Used by Biooptics with permission of Tanja Schwickert

9 DNA-content Ploidy 1n 2n 3n 4n dsdna dye Excitation Laser [nm] Emission [nm] Notes Sub-G1 G 1 /G 0 S M G2 PI Propidium iodide 7-AAD 7-Aminoactinomycin D 488/ dsdna/rna Fix/Perm 488/ dsdna Fix/Perm Cell-cycle DAPI 350/ dsdna Fix/Perm Hoechst / dsdna Cellmembrane permeable DNA-content (linear)

10 (alternative: Click-it EdU assay) G 1 /G 0 M G2 S-phase analysis by BrdU uptake In vitro/ in vivo Bromodeoxyuridine (BrdU) incorporation 42% Harvest cells/organ Time X Staining of surface antigen 4% Fixation/Permeablization DNase-treatment Staining of BrdU and total DNA BrdU-Fitc 7-AAD (DNA content) Bone Marrow: 30 min

11 Live/dead discrimination with impermeable dyes DNA dye Excitation [nm] Notes Live Dead PI Propidium iodide 7-AAD 7-Aminoactinomycin D 488/ /561 Non-fixable DAPI 350 Protein dye Excitation [nm] Notes LIVE/DEAD Fixable (Molecular Probes) Fixable viability dye efluor (ebioscience) 350/405/488/561/ /405/488/640 Fixable Zombie (BioLegend) 350/405/640 and many more Used by Biooptics with permission of Tanja Schwickert

12 Apoptosis Staining/dye method note Annexin 5 Phosphatidylserine (PS) translocation to outer membrane No Fix/Perm Ca2+ buffer dsdna dye: PI, 7-AAD, DAPI Membran impermeable DNA-fragmentation (Sub-G1) No Fix/Perm Fix/Perm Anti-pro-apoptotic molecules Active form of caspase-3 Cleaved PARP Fix/Perm JC-1 Mitochondrial Membrane Potential, Emission: 530nm (monomer in cytoplasm, apoptotic) 590nm (aggregates in mitochondria, live) No Fix/Perm ratiometric Live Apoptotic Dead Annexin V Dead PI PI/7-AAD Live Apoptotic Annexin V-Fitc

13 Proliferation Cell proliferation dyes Excitation [nm] CFSE 488 nonfluorescent ester amine-reactive fluorescent molecule (esterases activated) CellTrace Violet (Molecular Probes) efluor 450/670 (ebioscience) 405 VPD450 (BD) /640 and many more FSC-A IgG1 CellTrace Violet

14 Intracellular signaling: Ca 2+ -flux Calcium sensor dye Excitation Emission efluor nm 514nm Indo-1 346nm 410 nm (Ca 2+ ) 485nm Ca 2+ Ca 2+ Ca 2+ Ca 2+ Ca 2+ Ca 2+ Ca 2+ Flura-2 300nm (Ca 2+ ) 400nm 510nm Loading of cells with calcium sensor dye Igβ Ionomycin Incubation at 37 C Acquisition & Stimulation at 37 C Fortessa I (water bath ) efluor 514 Time

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