cellular techniques
DESCRIPTION
Studentpresentatie cellulaire technieken PT02TRANSCRIPT
Cellular techniques
G. Groenhof s0729884
S. Hemelaar s0729906
S. Hofstraat s0729914
K. Jenniskens s0729949
Y. Khaled s0729981
L. De Kroon s0730041
S. Van Kuijk s0625337
Properties used for separation
• Physical parameters
oSedimentation rate / size
oIntrinsic fluorescence
oBuoyant density
oAdherence
oAntibody binding
• Biological parameters
oUptake of particles
oMetabolic activity
FACS (Fluorescence Activated Cell Sorter)
• Separation by antibody affinity
• Fluorescent monoclonal
antibodies
• Flow cell
• Detection and charge
• Electric field
• Indirect FACS
• Flowcytometry
Immunofluorescence techniques
• Direct test
• Primary antibodies
• Indirect
• Secondary antibodies
• Sandwich
• Capture antibodies
• Secondary antibodies
Microscopy
• Light microscopy
• Histochemical colouring
• Electron microscopy
• Electron-dense immunolabel (colloidal gold)
• UV microscopy
• Fluorescence
Chromium release assay
• Ability of CTL to kill target cells (functional)
• Target cell labeled with Chromium 59 (intracellular)
• Cytotoxicity: release of radioactive protein into medium
• Repetition at different ratios
• Effector cells vs. Target cells
Limiting dilution analysis
• Estimate frequency of precursor cells
• Precursor cells: indication of immune response
• Cell suspension (~1 cell/ sample)
• Poisson distribution: 37% of samples contain no precursor cells
Plaque technique
• Agar gel coated with RBC
• Add complement
• B cells added
• Antibodies produced
• Lysis of RBC
• Clear spots on agar
• B cells in clear spots
ELISpot
• Immobilized antigen on well plate
• B cells added
• Produced antibodies bind to antigen
• Secondary antibody added
• Labeled with enzyme
• Coloured product solid in gel
Stadia of the cell cycle
• Interphase:
• G1 phase: increase cytoplasm, production proteins
• S phase: DNA replication
• G2 phase: production particles for mitosis
• Mitosis
• G0 phase: resting cell
Cell proliferation
• Properties for measuring
• Mitotic activity
• DNA activity
• Metabolic activity
• Cell proliferation measuring techniques
• Direct counting of cells
• Measuring 3H-thymidine, adding cytokines
• Scintillation counter
• Detecting dehydrogenase activity increase
• ELISA
T-cell proliferation test
Antigen Concentration5 μg 2.5 μg 1.25 μg 0 μg
A control
+ Antibiotic 1
20000
39500
9550
18505
4598
8500
800
750
B control
+ Antibiotic 2
1000
1200
25000
1250
10000
1400
900
150
C control
+ Antibiotic 3
7200
850
3500
800
1800
750
850
850
• T cells immunized with antigens A, B and C
• 3 different antigens in 4 different concentrations
• 3 different kinds of antibiotics
• Counting DNA-build-in radioactive Thymidine
T cells with antigen A
• T-cells respond adequately
• Antibiotic doubles proliferation (±)
oAntibiotic brakes down antigen -> APC activation -> More T-cell proliferation
oT-cell more sensitive
Antigen Concentration5 μg 2.5 μg 1.25 μg 0 μg
A control
+ Antibiotic 1
20000
39500
9550
18505
4598
8500
800
750
T cells with antigen B
• Antigen toxic to T-cell at high concentration (5 μg)
• At lower concentrations normal T-cell development
oAntibiotic neutralizes antigen
oAntibiotic destroys T-cells
Antigen Concentration5 μg 2.5 μg 1.25 μg 0 μg
B control
+ Antibiotic 2
1000
1200
25000
1250
10000
1400
900
150
T cells with antigen C
• T-cells respond adequately
• Antibiotic neutralizes antigen
Antigen Concentration5 μg 2.5 μg 1.25 μg 0 μg
C control
+ Antibiotic 3
7200
850
3500
800
1800
750
850
850