Cellular Techniques
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Transcript of Cellular Techniques
![Page 1: Cellular Techniques](https://reader035.fdocuments.net/reader035/viewer/2022062704/5560e41ad8b42a3d768b4ce4/html5/thumbnails/1.jpg)
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
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Properties used for separation
• Physical parameters
oSedimentation rate / size
oIntrinsic fluorescence
oBuoyant density
oAdherence
oAntibody binding
• Biological parameters
oUptake of particles
oMetabolic activity
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FACS (Fluorescence Activated Cell Sorter)
• Separation by antibody affinity
• Fluorescent monoclonal
antibodies
• Flow cell
• Detection and charge
• Electric field
• Indirect FACS
• Flowcytometry
![Page 4: Cellular Techniques](https://reader035.fdocuments.net/reader035/viewer/2022062704/5560e41ad8b42a3d768b4ce4/html5/thumbnails/4.jpg)
Immunofluorescence techniques
• Direct test
• Primary antibodies
• Indirect
• Secondary antibodies
• Sandwich
• Capture antibodies
• Secondary antibodies
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Microscopy
• Light microscopy
• Histochemical colouring
• Electron microscopy
• Electron-dense immunolabel (colloidal gold)
• UV microscopy
• Fluorescence
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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