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