W6L9 - Purification of Lymphocytes & Cell Based Immunoassays Flashcards
Ways to Purify Lymphocytes
Centrifugation - buffy coats - density gradients Magnetic bead separation Panning Flow Cytometry
Centrifugation - Buffy Coat
Centrifugation of whole blood will produce layers
- RBC, WBC and plasma layers
WBC layer is thin and called the buffy coat
Buffy coat can be removed by pipette to have concentrated WBC preparation
Not pure = RBC contamination
Centrifugation - Density Gradients
Separation based on sedimentation rates of cells
Sedimentation rate correlates to cell size
Different cells separated by use of substances having buoyancy densities between that of different cell types
After centrifugation cells will be layered according to density
Ficoll Hypaque Gradient
Most commonly used separating material
Mononuclear cells (lymphocytes, monocytes) layered on top of FHG
RBC and granulocytes layered under FHG
Platelets and plasma layered over mononuclear cell layer
Magnetic Bead Separation
Small magnetic beads coated with monoclonal antibodies
Added to blood and incubated
- specific cell type binds to beads forming rosettes
Magnet placed near tube and cell rosettes with beads migrate to magnet
Allows for other cells and serum to be removed
Panning
Monoclonal antibody coated on bottom of petri dish
Blood or cell suspension placed into petri dish to flood bottom
Incubation then decant fluid and wash
Specific cells trapped on bottom of dish by monoclonal Ab
Components of a Flow Cytometer
Fluidics - cell suspension in single file
Optics - generates light, collects light
Electronics - process optic signals
Flow Cytometry - Cell Surface Markers
When one antibody is paired with one dye to one marker a histogram can be produced
Determines numbers (%) of cell type
Can use two antibodies paired to two different dyes to two different markers in same tube
Scatter plot to determine % of cells for each individual as well as cells with dual marker
Flow Cytometry Sorting
Aka Fluorescent Activated Cell Sorting (FACS)
- specific antibodies labelled with fluorescent dyes reacted with cells
- cells passed through flow cytometer which identifies them
Cells become charged and can be separated by an electric field
Cell Based Assays
Tissue typing - Human Leukocyte Antigens (HLA) Functional Assays - Neutrophils - Lymphocyte proliferation - Cytotoxic T cell function
Why use HLA typing?
Transplantation Disease Association Forensics/paternity Anthropology Vaccine development
HLA
Cell surface proteins encoded by genes in the MHC
Inherited co-dominantly (paternally and maternally)
Belong to Ig superfamily of proteins
Serology Based Typing
Various names - microlymphocytotoxicity - complement dependent cytotoxicity - lymphocytotoxicity T cells used for class 1 (HLA - A, B, C) B cells used for class 2 (HLA - DR, DQ) Detects what is expressed on cell surface
How is Serology Based Typing done?
Serum containing known specific anti-HLA antibodies in tray wells
Lymphocytes added
- react with specific antibody if express specific HLA type
Rabbit complement added
- lymphocytes with Ag-Ab complex killed (enlarge)
Detect killing by addition of eosin dye
- killed cells larger and stained
- live cells smaller and refractile
Neutrophil Function Assays
Phagocytosis
- measure uptake of bacteria or latex particles by counting or label
Intracellular killing
- use Staph aureus - test for viability
Directional migration
Measure up-regulation of surface markers using monoclonal Abs
