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The flashcards below were created by user
J.Seet
on FreezingBlue Flashcards.
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What are four ways of purification of lymphocytes?
- Centrifugation (buffy coats, density gradients)
- Magnetic bead separation
- Panning
- Flow cytometry
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What is the buffy coat?
Thin layer of WBC after the centrifugation of whole blood
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Is the buffy coat pure?
- No
- will have RBC contamination
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What is the separation based on in density gradients?
sedimentation rates of cells
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What does sedimentation rate of cells correlate to?
cell size
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What are the (4) steps in magnetic bead separation?
- Small beads are coated with monoclonal antibodies
- Added to blood and incubated
- Magnet is placed near tube and cell rosettes with beads migrate to magnet
- Other cells and serum can be removed
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Why is CD8 used to collect lymphocytes by CD3 and CD4 aren't?
CD8 is used to collect lymphocytes because CD3 and CD4 are present in monocytes
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What is Panning? (4)
- Monoclonal antibody coated on bottom of petri dish
- Blood/cell suspension placed into petri dish to flood bottom
- Incubation then decant fluid & wash
- Specific cells trapped on bottom of petri dish by monoclonal antibody
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What is flow cytometry also known as?
flurenscent activated cell sorting (FACS)
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What is flow cytometry? (3)
- Specific antibodies labelled with fluorescent dyes react with blood/cell suspension
- Cells passed through flow cytometer which identifies labelled cells by the use of laser
- Cell becomes charged and can be separated by an electric field
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What are the 2 cell based assays?
- Tissue typing
- Functional assays
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What is involved in tissue typing?
human leucocyte antigen (HLA)
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What is involved in functional assays?
- Neutrophils
- Lymphocyte proliferation
- Cytotoxic T cell function
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Why do we use HLA typing? (5)
- Transplantation
- Disease association
- Forensic/Paternity
- Anthropology
- Vaccine development
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What is HLA?
- Human Leucocyte Antigen
- cell surface proteins encoded by genes within the MHC
- Inherited co-dominantly
- Belong to Ig super family of proteins
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What are the various names for serology based typing? (3)
- Mircolymphocytotoxicity
- Complement Dependent Cytotoxicity
- Lymphcytotoxicity
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T cells are used for what class?
class I
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What are the class I HLA?
HLA- A, B, C
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B cells are used for what class?
Class II
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What are the class II HLA?
HLA - DR, DQ
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What is Serology Based Typing?
- Serum contain known specific anti-HLA antibodies in tray wall
- Lymphocytes added to serum
- Rabbit complement added
- Detect killing by addition of eosin dye, ethridium bromide
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What is neutrophil function assays? (3)
- Phagocytosis
- Intracellular killing
- Directional migration
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What does neutrophil function assays measure?
Measure up-regulation of surface markers using monoclonal antibodies
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How does lymphocyte proliferation occur?
Lymphocytes stimulated by antigen/other activators and undergo cell division (in vitro)
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How is lymphocyte proliferation measured?
- Measuring release of cytokines by immunoassay (ELISA)
- Incorporation of radio labeled thymidine into DNA of dividing cells
- Incorporation of fluorescent dyes into plasma membrane of daughter cells from parent cell - measure by flow cytometry
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Cytotoxic T cells kill target cells. How is the function of cytotoxic T cell measured?
- Labelled with radioisotopes (51Cr)
- Mixed with lymphocytes and incubated
- Release 51Cr into surrounding media - measured T cell ability to kill target
- Similar assay used for NK cells
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