Cell based methods including flow cytometry Flashcards
what is cell based assay
is analysis based on cell culture method where live cells are grown and used as model to access biochemistry and physiology of love and dead cells.
what are common used methods in immunology
-isolation of cells include flow cytometry, magnetic beads
-apoptosis: trypan blue to visualize dead cells for cell viability
-microscope: cell counting, Immunohistochemistry IHC, immunofluorescence IF
-characterization of lymphocytes specificity and function: flow cytometry
what is a buffy coat
is a fraction of anticoagulated blood sample after centrifuge that contain white blood cells and platelets
how is leukocyte isolated from blood sample
by centrifugation of blood sample that separate red blood cell on the bottom and white blood cells and platelets on the top
what are the leukocytes types and their marker
- CD4+ T helper cells - CD3, CD4
- CD8+ T cytotoxic cells -CD3, CD8
- B cells : CD19
- NK cells- CD56
-macrophage - CD14
-dendritic cells
-basophils
what are two method of isolating cells with magnetic beads
- direct labeling: is when specific antibody is labeled directly with microbeads /magnetic beads. occurs in one step
- indirect labeling: first there is primary antibody that binds to the antigen cell surface and microbeads will bind to primary antibody. occurs in two steps
what are different ways of indirect cells labelling
- antibody microbeads
-streptadin microbeads
-biotinylated microbeaads
-fluorochrome conjugated antibody
what is positive and negative isolation their pros and cons
positive isolation: is labeling of wanted/target cells
pros; maximize purity and reduce washing steps
cons; activate target cells
negative isolation: is labeling of unwanted cells
pros; easy removal of unwanted cells
cons; had to yield purity for small targeted population
what is the separate technique of MACS cells
- for positive selection: targeted cells are labeled with MACs microbeads are are separated in MACs column where labeled cells are collected as negative fraction and they will elute in the column and unlabeled cells are retains as positive fraction
- for negative selection; un targeted cells are labeled with biotinylated antibody and anti-biotin microbeads, and un target cells are retained in the column as the targeted cells passes the column and are collected as enriched positive fraction
what do we use to evaluate cell viability and cell counting
we can use trypan blue which stain dead cells and we will be able to visualize it
what is IHC
is the use of antibody labeled as specific agent through antibody-antigen interactions
how is IHC used or perform its function
here antibody is chemically attached to the enzyme that can covert colourless substrate into coloured reaction product. the coloured product can be visualized in light microscope
what is IF
IF is used to visualize protein in the tissues using antibody that is conjugated to the fluorescent dye.
explain direct and indirect of IF
direct: is when primary antibody is directly conjugated to fluorescent dye and
indirect is when secondary that is conjugated with fluorescent dye is attached to primary antibody binding to cell surface of antigen for visualization of protein in the tissue
what colour is B cells and HLA-DR are visualized
B cells is red and HLA-DR is green
Characterisation of lymphocyte specificity and function
cell growth/ proliferate
what are different ways to evaluate cell growth
-H-thymidine
- cell trace staining : we use flow cytometry
why is H-thymidine is incorporated into DNA when cells divide
because it has high level of cell division
what does it mean to have high level of radioactivity
means they have high level of proliferation
how do we analyze the lymphocyte characterization using flow cytometry
by staining cells with cell trace stain
what is ELISPOT assay
-is a simple tool to measure T cells responses
it can be use to know how many cytokine secreted by T cells. and also how many antibody was secreted by B cells
how do we analyze if T cells produce cytokines by using ELISPOT assay
- we first add antibody specific in binding cytokines
- activated T cells are added of different effector function
- some activated T cells will produce cytokines which will bind to the specific antibody
- and bound cytokines are revealed with secondary antibody that is conjugated to the enzyme
is spotted by giving insoluble precipitate
how can you cunt number of Tcells secreted cytokine
-first add same amount of T cells in the control wells and other wells
-add peptide in other wells
-patient T cells will respond to the peptide by secreting cytokines
- because we know how many T cells we have we can know how many T cells secreted cytokines
how is ELISPOT used to know how many antibody was secreted by B cells
by using specific antigen coated on the surface and enzyme labeled antibody
