Download Detection of Intracellular proteins

Detection of intracellular antigens
at the single cell level using flow
cytometry
Niga Nawroly
Imperial College London
[email protected]
HOW?
Surface Phenotyping
+
Intracellular
+
How does a flow cytometer work?
Electronics
Fluidics
Light detectors
FL3 PerCP, Cy5
FL2 PE
FL1 FITC
SSC
Laser
Light
488nm
FSC
Computer
Detection of Intracellular proteins:
Studying Intracellular Enzyme Activity
• Enzymes are found in all cell types.
• Involved in almost every cellular process.
• Cyclooxygenase (COX)
• Terminal Deoxynucleotidyl Transferase (TdT)
• Myeloperoxidase (MPO)
Fixation does not allow the detection of active enzyme in a cell.
Detection of Intracellular proteins:
Signal transduction
Important in distinguishing changes in signaling status that
arise in cells during functional activation.
• Phosphorylated STAT-1
• KINASES (p38 MAPK,P44/42 MAPK, JNK/SAP).
• Members of cell survival pathways (AKT/PKB)
• T cell activation pathway (TYK2)
• Anti Phosphorylated ERK
Detection of Intracellular proteins:
Immunological responses
Inflammatory mediators such as perforin or granzyme.
Cytokines and Chemokines
Cytokines
Cytokines are small secreted proteins produced by
lymphocytes.
• Mediate and regulate immunity, inflammation, and
hematopoiesis.
• Produced in response to an immune stimulus.
• They bind to specific membrane receptors.
Cytokine Production
Signal
Cytokines
Picture From www.fredonia.edu
LPS
Ionomycin
PHA
T cell
Con A
CD3
CD28
To enhance the accumulation of intracellular cytokines.
Stimulation
Monensin: Cytokines accumulate in the ER
Brefeldin A: in Golgi complex.
Secretion stop
(Brefeldin A or Monensin)
• Polyclonal stimulation
Only in vitro
• Specific, monoclonal stimulation…
Peptide, Virus, Bacteria
To maintain structural integrity.
Formaldehyde or glutaraldehyde
Keep the protein structure and doesn't change the
Saponin
(permeablisation
buffer).
(accessibility
of the) epitopes
too much
Intracellular Staining
Permeabilisation
Fixation
Multiparameter Intracellular technique
Two or more antibodies!...
To analyse multiparameter data at the level of one cell
• Cell size and granularity
• CD antigens
• Cytokine receptors
• Adhesion molecules
• Migration/Homing molecules
• Cell cycle, G0, G1, S, G2
Combination of surface
and intracellular
flow cytometry.
1 Colour
2 Colour
Advantages of Multi-colour
4
2
1
3 Colours
2 Population
12
2
3
3
1
4 Colours
5 Colours
6 Colours
1
2
24
40
60
?
Which fluorochromes and when …
ANTIGEN DENSITY
FLUOROCHROME
low
Phycoerythrin, APC
low-intermediate
CY5
high
FITC, PerCP
What dye?
Problems
Non-specific binding (false +Ve result)
Fc receptor blocking,
Mouse systems, FcγII/III CD16/CD32
Human, human Ig or 10% autologous serum in PBS
Fc Receptor
Anti-Fc Receptor
Problems
Non-specific binding (false +Ve result)
Immunoglobulin Isotype Controls
Same fluorochrome-conjugated antibody of
irrelevant specificity which has the same Ig isotype.
Ligand blocking control
Pre-block with anti-cytokine antibody.
Problems
Autofluorescence
Run an unstained sample
Dead cells
Centrifugation, Ficoll sepaeration, or by using propidium iodide.
Can be detected on Forward Scatter channel….
Spectral Overlap
Single-stained cells. Apply electronic compensation
Problems
Spectral overlap
FL-2
PMT
FL-1
PMT
FL-1 -% FL-2
FL-2 -% FL-1
Electronic compensation
10 4
10
11
PE
10 2
PE
10 2
10 3
10 4
11
10 3
10
R10
CD8
10 1
10 1
R10
12
13
10 1
10 2
APC
10 3
10 4
RANTES
Lung BALB/c
Jonathan Dodd
12
13
10 1
10 2
APC
10 3
10 4
4
10
4
15
10
11
IL-5-APC
10 2
CD3-QR
10 2
10
10
3
3
10
14
CD3
16
R10
17
10
1
2
10
DX5-PE
10
3
10
10
IL-5
R14
1
1
NK T
10
12
4
13
10
1
INF-g
DX5
BAL BALB/c
Wieslawa Olszewska
10 2
IFNg-FITC
10
3
10
4
Data Analysis
Available analysis software
• Cellquest
• WinList
• Flowjo
• WinMDI
Free cytometry software:
http://flowcyt.cyto.purdue.edu/flowcyt/software/Catalog.htm
Flow Cytometry on the web
• ISAC www.isac-net.org
• Salk Institute http://flowcyt.salk.edu
• Purdue University www.cyto.purdue.edu
• Scripps Research Institute http://facs.scripps.edu/index.html
• CRUK http://science.cancerresearchuk.org/sci/facs
Our website
http://www.wfi.med.ic.ac.uk/Flow.htm
New Website address:
www1.imperial.ac.uk/medicine/about/institutes/wfi/flow
Our facility provides:
• BD FACS LSR machine (analyser).
Three-laser, Six-colour benchtop flow cytometer.
• BD FACS Calibur machine (analyser).
One laser (488-nm) four-colour
multipurpose flow cytometer.
• BD FACSDiva machine (cell sorter).
Three-laser, Six-colour cell sorter.
Our facility provides:
• Luminex Xmap, multi parameter array.
• ELISPOT reader, to measure AntigenSpecific Cytokine Production of Single Cells.
plus stand-alone analysis computers that can
be used for off-line analysis.
www1.imperial.ac.uk/medicine/about/institutes/wfi/flow
[email protected]