introduction to flow cytometry - university of pittsburgh · what is flow cytometry? •single-cell...
TRANSCRIPT
![Page 1: Introduction to Flow Cytometry - University of Pittsburgh · What is Flow Cytometry? •Single-cell analysis. •Uses monoclonal antibodies to tag markers on/inside the cells. •Fluorescent](https://reader035.vdocuments.us/reader035/viewer/2022081504/5ede037dad6a402d66694653/html5/thumbnails/1.jpg)
Introduction to Flow Cytometry
Aarika MacIntyreSenior Flow Core Technologist
![Page 2: Introduction to Flow Cytometry - University of Pittsburgh · What is Flow Cytometry? •Single-cell analysis. •Uses monoclonal antibodies to tag markers on/inside the cells. •Fluorescent](https://reader035.vdocuments.us/reader035/viewer/2022081504/5ede037dad6a402d66694653/html5/thumbnails/2.jpg)
What is Flow Cytometry?
• Single-cell analysis.
• Uses monoclonal antibodies to tag markers on/inside the cells.
• Fluorescent molecules (flurochromes) are bound to the antibodies.
• Flurochromes are excited by lasers at specific wavelengths.
• Fluorochromes emit light at a higher wavelength, which is read by the cytometer.
![Page 3: Introduction to Flow Cytometry - University of Pittsburgh · What is Flow Cytometry? •Single-cell analysis. •Uses monoclonal antibodies to tag markers on/inside the cells. •Fluorescent](https://reader035.vdocuments.us/reader035/viewer/2022081504/5ede037dad6a402d66694653/html5/thumbnails/3.jpg)
Basics of Flow – Fluidics
Sheath Fluid Sample
Flow Cell (Hydrodynamic Focusing)
Fluorescence
FSC/SSC
Laser
![Page 4: Introduction to Flow Cytometry - University of Pittsburgh · What is Flow Cytometry? •Single-cell analysis. •Uses monoclonal antibodies to tag markers on/inside the cells. •Fluorescent](https://reader035.vdocuments.us/reader035/viewer/2022081504/5ede037dad6a402d66694653/html5/thumbnails/4.jpg)
Basics of Flow - Fluidics
![Page 5: Introduction to Flow Cytometry - University of Pittsburgh · What is Flow Cytometry? •Single-cell analysis. •Uses monoclonal antibodies to tag markers on/inside the cells. •Fluorescent](https://reader035.vdocuments.us/reader035/viewer/2022081504/5ede037dad6a402d66694653/html5/thumbnails/5.jpg)
Basics of Flow - Fluidics
![Page 6: Introduction to Flow Cytometry - University of Pittsburgh · What is Flow Cytometry? •Single-cell analysis. •Uses monoclonal antibodies to tag markers on/inside the cells. •Fluorescent](https://reader035.vdocuments.us/reader035/viewer/2022081504/5ede037dad6a402d66694653/html5/thumbnails/6.jpg)
Basics of Flow
FSCSS
C
Mammal whole blood/bone marrow
![Page 7: Introduction to Flow Cytometry - University of Pittsburgh · What is Flow Cytometry? •Single-cell analysis. •Uses monoclonal antibodies to tag markers on/inside the cells. •Fluorescent](https://reader035.vdocuments.us/reader035/viewer/2022081504/5ede037dad6a402d66694653/html5/thumbnails/7.jpg)
Basics of Flow
Time
Inte
nsi
ty
A
H
W
![Page 8: Introduction to Flow Cytometry - University of Pittsburgh · What is Flow Cytometry? •Single-cell analysis. •Uses monoclonal antibodies to tag markers on/inside the cells. •Fluorescent](https://reader035.vdocuments.us/reader035/viewer/2022081504/5ede037dad6a402d66694653/html5/thumbnails/8.jpg)
Basics of Flow
![Page 9: Introduction to Flow Cytometry - University of Pittsburgh · What is Flow Cytometry? •Single-cell analysis. •Uses monoclonal antibodies to tag markers on/inside the cells. •Fluorescent](https://reader035.vdocuments.us/reader035/viewer/2022081504/5ede037dad6a402d66694653/html5/thumbnails/9.jpg)
Fluorochromes
![Page 10: Introduction to Flow Cytometry - University of Pittsburgh · What is Flow Cytometry? •Single-cell analysis. •Uses monoclonal antibodies to tag markers on/inside the cells. •Fluorescent](https://reader035.vdocuments.us/reader035/viewer/2022081504/5ede037dad6a402d66694653/html5/thumbnails/10.jpg)
![Page 11: Introduction to Flow Cytometry - University of Pittsburgh · What is Flow Cytometry? •Single-cell analysis. •Uses monoclonal antibodies to tag markers on/inside the cells. •Fluorescent](https://reader035.vdocuments.us/reader035/viewer/2022081504/5ede037dad6a402d66694653/html5/thumbnails/11.jpg)
Fluorochromes• Many “colors” to choose from.
• Each fluorochrome has two properties: excitation and emission.
• Excitation: wavelength at which the fluorochrome absorbs the most energy.
• Emission: wavelength at which the fluorochrome produces the most energy.
![Page 12: Introduction to Flow Cytometry - University of Pittsburgh · What is Flow Cytometry? •Single-cell analysis. •Uses monoclonal antibodies to tag markers on/inside the cells. •Fluorescent](https://reader035.vdocuments.us/reader035/viewer/2022081504/5ede037dad6a402d66694653/html5/thumbnails/12.jpg)
Fluorochromes
![Page 13: Introduction to Flow Cytometry - University of Pittsburgh · What is Flow Cytometry? •Single-cell analysis. •Uses monoclonal antibodies to tag markers on/inside the cells. •Fluorescent](https://reader035.vdocuments.us/reader035/viewer/2022081504/5ede037dad6a402d66694653/html5/thumbnails/13.jpg)
Fluorochromes
• Fluorescent proteins• GFP, YPF, CFP, RFP
• Viability Dyes• DNA Dyes – DAPI, PI, 7AAD, etc.
• Fixable Viability Dyes
![Page 14: Introduction to Flow Cytometry - University of Pittsburgh · What is Flow Cytometry? •Single-cell analysis. •Uses monoclonal antibodies to tag markers on/inside the cells. •Fluorescent](https://reader035.vdocuments.us/reader035/viewer/2022081504/5ede037dad6a402d66694653/html5/thumbnails/14.jpg)
![Page 15: Introduction to Flow Cytometry - University of Pittsburgh · What is Flow Cytometry? •Single-cell analysis. •Uses monoclonal antibodies to tag markers on/inside the cells. •Fluorescent](https://reader035.vdocuments.us/reader035/viewer/2022081504/5ede037dad6a402d66694653/html5/thumbnails/15.jpg)
Compensation
![Page 16: Introduction to Flow Cytometry - University of Pittsburgh · What is Flow Cytometry? •Single-cell analysis. •Uses monoclonal antibodies to tag markers on/inside the cells. •Fluorescent](https://reader035.vdocuments.us/reader035/viewer/2022081504/5ede037dad6a402d66694653/html5/thumbnails/16.jpg)
Compensation
![Page 17: Introduction to Flow Cytometry - University of Pittsburgh · What is Flow Cytometry? •Single-cell analysis. •Uses monoclonal antibodies to tag markers on/inside the cells. •Fluorescent](https://reader035.vdocuments.us/reader035/viewer/2022081504/5ede037dad6a402d66694653/html5/thumbnails/17.jpg)
• Single-stained controls are required; one for each fluorochrome.• Cells
• Beads
• Unstained control also necessary. • Cells are ideal
• Software will calculate the percent
overlap for each color.
Compensation
Un
stai
ne
d C
ells
FITC
on
ly
PE
on
ly
Pe
rCP
on
ly
Fully
Sta
ined
Sam
ple
![Page 18: Introduction to Flow Cytometry - University of Pittsburgh · What is Flow Cytometry? •Single-cell analysis. •Uses monoclonal antibodies to tag markers on/inside the cells. •Fluorescent](https://reader035.vdocuments.us/reader035/viewer/2022081504/5ede037dad6a402d66694653/html5/thumbnails/18.jpg)
Compensation
![Page 19: Introduction to Flow Cytometry - University of Pittsburgh · What is Flow Cytometry? •Single-cell analysis. •Uses monoclonal antibodies to tag markers on/inside the cells. •Fluorescent](https://reader035.vdocuments.us/reader035/viewer/2022081504/5ede037dad6a402d66694653/html5/thumbnails/19.jpg)
• Compensation problems usually caused by poor panel design.
• In general, if two fluorochromes:a) are excited by the same laser, and
b) use the same filter for detection, then
they cannot be run together on a conventional cytometer.
Compensation
![Page 20: Introduction to Flow Cytometry - University of Pittsburgh · What is Flow Cytometry? •Single-cell analysis. •Uses monoclonal antibodies to tag markers on/inside the cells. •Fluorescent](https://reader035.vdocuments.us/reader035/viewer/2022081504/5ede037dad6a402d66694653/html5/thumbnails/20.jpg)
Compensation
![Page 21: Introduction to Flow Cytometry - University of Pittsburgh · What is Flow Cytometry? •Single-cell analysis. •Uses monoclonal antibodies to tag markers on/inside the cells. •Fluorescent](https://reader035.vdocuments.us/reader035/viewer/2022081504/5ede037dad6a402d66694653/html5/thumbnails/21.jpg)
• Sometimes colors that should work together don’t.
Compensation
![Page 22: Introduction to Flow Cytometry - University of Pittsburgh · What is Flow Cytometry? •Single-cell analysis. •Uses monoclonal antibodies to tag markers on/inside the cells. •Fluorescent](https://reader035.vdocuments.us/reader035/viewer/2022081504/5ede037dad6a402d66694653/html5/thumbnails/22.jpg)
• Spread color choices across available excitation lasers.
•Choose colors with emissions as far apart as possible.
•Match brighter colors with dimmer markers.• Some colors fluoresce brighter than others.
• Some markers are less frequently found on the cell surface.
• Come to Heidi’s presentation to learn more!
Panel Design
![Page 23: Introduction to Flow Cytometry - University of Pittsburgh · What is Flow Cytometry? •Single-cell analysis. •Uses monoclonal antibodies to tag markers on/inside the cells. •Fluorescent](https://reader035.vdocuments.us/reader035/viewer/2022081504/5ede037dad6a402d66694653/html5/thumbnails/23.jpg)
Panel Design
355nm UV
647nm Red
488nm Blue
405nm Violet
532nm Y/G
![Page 24: Introduction to Flow Cytometry - University of Pittsburgh · What is Flow Cytometry? •Single-cell analysis. •Uses monoclonal antibodies to tag markers on/inside the cells. •Fluorescent](https://reader035.vdocuments.us/reader035/viewer/2022081504/5ede037dad6a402d66694653/html5/thumbnails/24.jpg)
![Page 25: Introduction to Flow Cytometry - University of Pittsburgh · What is Flow Cytometry? •Single-cell analysis. •Uses monoclonal antibodies to tag markers on/inside the cells. •Fluorescent](https://reader035.vdocuments.us/reader035/viewer/2022081504/5ede037dad6a402d66694653/html5/thumbnails/25.jpg)
• Single-cell suspension
• 106 cells/mL, minimum 250µL
• 12x75 Polystyrene tube
• Filter at the instrument – bring a pipette!
For help with a staining protocol or tissue prep, see Ailing or other members of the Flow Core.
Sample Prep
![Page 26: Introduction to Flow Cytometry - University of Pittsburgh · What is Flow Cytometry? •Single-cell analysis. •Uses monoclonal antibodies to tag markers on/inside the cells. •Fluorescent](https://reader035.vdocuments.us/reader035/viewer/2022081504/5ede037dad6a402d66694653/html5/thumbnails/26.jpg)
Dewayne Falkner – Operational Director [email protected]
Aarika MacIntyre – Senior Flow Core Technologist [email protected]
Ailing Liu – Research Specialist [email protected]
Heidi Gunzelman – Senior Flow Core Tech [email protected]
Nan Sheng – Flow Core Tech [email protected]
Thank you!