LYMPHOCYTE PURIFICATION & CELL BASED ASSAYS

what are 4 methods of lymphocyte purification

centrifugation

magnetic bead separation

panning

flow cytometry

what are the 3 layers produced by blood centrifugation

red blood cell layer

white blood cell layer

plasma layer

what is the buffy coat

the thin white blood cell layer produced in centrifugation

explain density gradients

it is the use of substances that have densities between those of the layers produced

so upon centrifugation the substances provide better separation between the blood component layers

explain separation when Ficoll Hypaque is used

Mononuclear cells layer on top of the ficoll hypaque gradient

rbc and granulocytes layer beneath the ficoll hypaque gradient

platelets and plasma layered over the mononuclear cells

explain separation using small magnetic beads

small magentic beads are coated in monoclonal antibodies

these beads are then placed in blood, and as it incubates the target substance binds to the antibodies

by placing a magnet near the tube the beads bound to the target will migrate wherever the magnet is moved

positively isolated cells are those removed by the magnet

negatively isolated cells are those left in the supernatant

explain separation by panning

monoclonal antibodies coat the bottom of a petri dish

a blood suspension is placed into the petri dish to flood the bottom

petri dish is incubated, decanted and washed

specific cells will be trapped to the bottom of the petri dish by the monoclonal antibodies

list the components of a flow cytometer

fluidics → cell suspension in single file

optics → generates light and collects light

electronics → processes optic signals

explain how a flow cytometer works

cells labelled with conjugate antibodies, with a fluorescent tag, are interrogated by laser

the flow cytometer measures the forward and side scatter, as well as which fluorescent label was present

what does each scatter represent

forward scatter is cell size

side scatter is cell granularity

how is leukaemia now diagnosed in reference to cell assays

diagnosed using cell surface markers

different leukaemia types have different expression of cell surface markers

panels of monoclonal antibodies are used to detect the cell surface proteins, and flow cytometry can do this

how can flow cytometry sort cells

it is done by fluorescent activated cell sorting

labelled cells are identified by the flow cytometer lasers

these cells also become charged and can be separated by an electric field

name a tissue typing assay

human leukocyte antigens (HLA)

explain how serology based HLA typing works

serum with known anti-HLA antibodies is in tray wells

lymphocytes are added to the serum and if correct HLA present, it’ll bind to the antibodies.

then a rabbit complement is added, which kills lymphocytes with the antigen-antibody complex

killing is detected with eosin stain; killed cells stained and larger, live cells smaller and refractile

which HLA is associated with T cells/MHC Class I

HLA-A,B,C

which HLA is associated with B cells/MHC Class II

HLA-DR,DQ

briefly describe the 4 neutrophil function assays

phagocytosis → measure bacteria or latex particle uptake by counting or label

intracellular killing → use staph. aureus

directional migration → movement through filters or gradients

measure up-regulation of surface markers using monoclonal antibodies

how can lymphocyte proliferation be measured

using ELISA’s to measure cytokine release

incorporate radiolabelled thymidine into dividing cell DNA
incorprate fluorescent dye into plasma membrane of daughter cells from parent cell (daughter cells have ½ of parent cell dye content) and measure with flow cytometry

how can cytotoxic T cell function be measured

target cell is labelled with Cr51 radioisotope

then this is mixed in with T lymphcytes and incubated

Cr51 releases into the surrounding media, and the amount of release is the measure of the T cell’s ability to kill the target