Flow cytometry, ELISA, ELISPOT & cell culture methods Sanjaya Adikari Department of Anatomy
 
Flow cytometer <ul><li>A nalyze suspended individual cells (or particles) </li></ul><ul><li>C ells flow through one or mor...
Laser beam
Stain T cells with Anti –CD3-PE ab   and Anti-CD4-FITC ab FITC PE Control FITC PE CD3 CD8 CD4 CD3 + CD3 + CD4 +
 
Enzyme-Linked Immunosorbent Assay  (ELISA) 96-well plate
Main steps                                                                         Patient serum which contains antibody A...
 
Positive ELISA Test Negative ELISA Test
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 ...
OD Concentration (ng/pg) sample
Enzyme-Linked Immunosorbent  Spot   ELISPOT                                                       Coat the plates with  an...
Examples of images from the ELISpot technique: Production of Interferon-gamma by human Peripheral Blood Mononuclear Cells ...
Tissue Culture Methods Tissue culture  organ culture  cell culture  p rimary cell cultures  continuous cell lines
<ul><li>Laminar flow hood </li></ul><ul><li>CO 2  incubator </li></ul><ul><li>Inverted microscope </li></ul><ul><li>Pipett...
Reagents <ul><li>Culture m edium   </li></ul><ul><ul><li>Bulk ions  - Na, K, Ca, Mg, Cl, P, Bicarb or CO 2 Trace elements ...
Procedure for MNC culture lymphoprep Peripheral blood MNCs RBC Neutrophils Density gradient centrifugation
 
<ul><li>Cells suspended in complete medium </li></ul><ul><li>T 37C ,  CO2 5-10%,   pH 7.2-7.5 ,  humid & dark </li></ul><u...
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Flowcytometer & elisa

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Flowcytometer & elisa

  1. 1. Flow cytometry, ELISA, ELISPOT & cell culture methods Sanjaya Adikari Department of Anatomy
  2. 3. Flow cytometer <ul><li>A nalyze suspended individual cells (or particles) </li></ul><ul><li>C ells flow through one or more lasers </li></ul><ul><li>Light scatter provides some information regarding the morphology of the particles </li></ul><ul><li>fluorescent dyes to label different cellular molecules or structures, such as membrane antigens, DNA, intracellular enzymes, ions, or organelles </li></ul>
  3. 4. Laser beam
  4. 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 +
  5. 7. Enzyme-Linked Immunosorbent Assay (ELISA) 96-well plate
  6. 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
  7. 10. Positive ELISA Test Negative ELISA Test
  8. 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
  9. 12. OD Concentration (ng/pg) sample
  10. 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
  11. 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)                                                                                                   
  12. 15. Tissue Culture Methods Tissue culture organ culture cell culture p rimary cell cultures continuous cell lines
  13. 16. <ul><li>Laminar flow hood </li></ul><ul><li>CO 2 incubator </li></ul><ul><li>Inverted microscope </li></ul><ul><li>Pipettes </li></ul>Work area and E quipment
  14. 17. Reagents <ul><li>Culture m edium </li></ul><ul><ul><li>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 </li></ul></ul><ul><li>Antibiotics </li></ul><ul><li>Buffers </li></ul><ul><li>Cytokines - GM-CSF, M-CSF, IL-4, IL-2 </li></ul>
  15. 18. Procedure for MNC culture lymphoprep Peripheral blood MNCs RBC Neutrophils Density gradient centrifugation
  16. 20. <ul><li>Cells suspended in complete medium </li></ul><ul><li>T 37C , CO2 5-10%, pH 7.2-7.5 , humid & dark </li></ul><ul><li>Visually inspect daily </li></ul><ul><li>Periods outside incubator minimal </li></ul><ul><li>Feeding with fresh medium every 3 - 5 days </li></ul><ul><li>Harvesting ( Mechanical , Proteolytic enzymes , EDTA ) </li></ul>Procedure ……. Cont..
  17. 21. DC

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