W6L9 - Purification of Lymphocytes & Cell Based Immunoassays Flashcards

1
Q

Ways to Purify Lymphocytes

A
Centrifugation
- buffy coats
- density gradients
Magnetic bead separation
Panning
Flow Cytometry
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2
Q

Centrifugation - Buffy Coat

A

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

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3
Q

Centrifugation - Density Gradients

A

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

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4
Q

Ficoll Hypaque Gradient

A

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

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5
Q

Magnetic Bead Separation

A

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

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6
Q

Panning

A

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

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7
Q

Components of a Flow Cytometer

A

Fluidics - cell suspension in single file
Optics - generates light, collects light
Electronics - process optic signals

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8
Q

Flow Cytometry - Cell Surface Markers

A

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

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9
Q

Flow Cytometry Sorting

A

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

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10
Q

Cell Based Assays

A
Tissue typing
- Human Leukocyte Antigens (HLA)
Functional Assays
- Neutrophils
- Lymphocyte proliferation
- Cytotoxic T cell function
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11
Q

Why use HLA typing?

A
Transplantation
Disease Association
Forensics/paternity 
Anthropology 
Vaccine development
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12
Q

HLA

A

Cell surface proteins encoded by genes in the MHC
Inherited co-dominantly (paternally and maternally)
Belong to Ig superfamily of proteins

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13
Q

Serology Based Typing

A
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
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14
Q

How is Serology Based Typing done?

A

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

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15
Q

Neutrophil Function Assays

A

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

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16
Q

Lymphocyte Proliferation

A

Lymphocytes stimulated by antigen or other activators undergo cell division
Measured by:
- measuring release of cytokines by ELISA
- incorporation of radiolabeled thymidine into DNA of dividing cells
- incorporation of fluorescent dyes into plasma membrane of daughter cells from parent cell

17
Q

Cytotoxic T cell Function

A

Measure of how well cytotoxic T cells can kill target cell

  • target cells labelled with radioisotope (51Cr)
  • mixed with lymphocytes and incubated
  • release of 51Cr into surrounding media is a measure of T cells ability to kill target