The document describes several immunology techniques: Flow cytometry analyzes individual cells or particles using laser light scattering and fluorescent dyes to label cellular molecules. ELISA uses antigen-coated plates and enzyme-linked antibodies to detect antibodies in serum samples. ELISPOT counts antibody-secreting cells by detecting individual antibody spots. Tissue culture methods grow cells in controlled conditions using culture medium, reagents, and equipment to maintain cells outside the body.

1. Flow cytometry, ELISA, ELISPOT & cell culture methods Sanjaya Adikari Department of Anatomy

3. Flow cytometer A nalyze suspended individual cells (or particles) C ells flow through one or more lasers Light scatter provides some information regarding the morphology of the particles fluorescent dyes to label different cellular molecules or structures, such as membrane antigens, DNA, intracellular enzymes, ions, or organelles

4. Laser beam

5. Stain T cells with Anti –CD3-PE ab and Anti-CD4-FITC ab FITC PE Control FITC PE CD3 CD8 CD4 CD3 + CD3 + CD4 +

7. Enzyme-Linked Immunosorbent Assay (ELISA) 96-well plate

8. Main steps Patient serum which contains antibody Anti-human immunoglobulin coupled to an enzyme. This is the second antibody, and it binds to human antibodies. Chromogen or substrate which changes colour when cleaved by the enzyme attached to the second antibody Coat the plates with antigen

10. Positive ELISA Test Negative ELISA Test

11. The cut-off value indicating a positive result is 0.500 O D of 0.300 to 0.499 are indeterminate and need to be retested Values < 0.300 are considered to be negative (+) Control (-) Control Patient A Patient B Patient C Assay Control 1.689 0.153 O.055 0.412 1.999 0.123

12. OD Concentration (ng/pg) sample

13. Enzyme-Linked Immunosorbent Spot ELISPOT Coat the plates with antigens Cells Anti-human immunoglobulin coupled to an enzyme. This is the second antibody, and it binds to human antibodies. Chromogen or substrate which changes color when cleaved by the enzyme attached to the second antibody Cells secrete antibodies

14. Examples of images from the ELISpot technique: Production of Interferon-gamma by human Peripheral Blood Mononuclear Cells (PBMC): Interferon-gamma (IFN-  ) Human unstimulated (250.000 cells) PPD stimulation (250.000 cells) TT stimulation (250.000 cells)

15. Tissue Culture Methods Tissue culture organ culture cell culture p rimary cell cultures continuous cell lines

16. Laminar flow hood CO 2 incubator Inverted microscope Pipettes Work area and E quipment

17. Reagents Culture m edium Bulk ions - Na, K, Ca, Mg, Cl, P, Bicarb or CO 2 Trace elements - iron, zinc, selenium S ugars - glucose is the most common A mino acids - 13 essential V itamins - B, etc. C holine, inositol S erum Antibiotics Buffers Cytokines - GM-CSF, M-CSF, IL-4, IL-2

18. Procedure for MNC culture lymphoprep Peripheral blood MNCs RBC Neutrophils Density gradient centrifugation

20. Cells suspended in complete medium T 37C , CO2 5-10%, pH 7.2-7.5 , humid & dark Visually inspect daily Periods outside incubator minimal Feeding with fresh medium every 3 - 5 days Harvesting ( Mechanical , Proteolytic enzymes , EDTA ) Procedure ……. Cont..

21. DC