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
what is cytometry
refers to measurement of physical and chemical characteristics of the cells.
example: cell marker, cell size and shape, cytokines production
what is flow cytometry
is when such measurements are make when cells are passing through measuring apparatus
what is flow sorting
extends flow cytometry by using electrical or chemicals means
what is the application of flow cytometry
cell counting, cell staining
what does flow cytometry tell us about cells
- its granularity
-its relative size
allow investigation of cell structure and function
How is a dot plot generated?
dead cells
variable cells
apoptotic cells: cells die when they achieve maximum cell division and can no longer reproduce
what is fluorochrome
are component of molecule that absorb light and emit fluoroscence. they contain aromatic group
what is the importance of labeling antibody with fluorochrome
this can be used to mark cell surface of antigen
How is a dotplot generated btn PE and FITC
- positive PE generation and positive FITC generation
- negative generation marking dead cells
- double positive PE and FITC generation
what does dotplot interpretation show us (see note slide 38)
it can show us how many percentage of cells present in different generation
What is the viability status of marked cell populations?
-cells that are both negative for annexin V and 7AAD has no viability or apoptosis, means that PS translocation has not occurred and plasma membrane is still intact.
- cells that are annexin V positive and 7AAD negative means that they are in early apoptosis but cell membrane are still intact
- cells that are double positive for annexin V and 7AAD means cell death or last stage of apoptosis, the cell membrane is not intact
what are important factor for flow cytometry
- compensation: is needed when we are using multiple fluorophores whose emission signal overlap
- unspecific binding: can be due to Fc receptor binding, where specific binding is for high affinity and saturated cells and unspecific binding is for low affinity and unsaturated
- isotype control: use antibody of the same isotype as marker of the interest but from different species
- autofluorescence: know that cell fluorescent even if there is no satining added, autofluoroscent come from normal cell component including riboflavin and flavoprotein
-multiplex analysis: analyzing using gate or subgate
application of flow cytometry
-allow cell separation of the interest from heterogenous cell population
how do you Analysis secretion of cytokines by Cytokine Bead Array
- sample is added to capture beads mix
- labeled antibody that is specific to the molecule/ primary antibody are added
- then captured analyte are analyzed by using PE-conjugated antibody
summary must know
- leukocytes can be isolated by density gradient centrifugation, magnetic beads (MACS, Dynabeads) and
flow cytometric cell sorting. - Microscopy can be used to count cells (trypan blue) and to assess phenotype and localization of cells in
tissue sections by immunofluorescence and immunohistochemistry. Number of cells analysed fairly
limited. Traditionally, few markers analysed. More extensive multiplexing only recently developed. - Flow cytometry can be used for analysis of cells in suspension. Multi-parameter analysis is and has for
long been common (up to 20-30 markers in conventional flow cytometry) and thousands of cells can be analysed per second. Gating strategies can be applied for detailed analysis of specific cell types. Spectralinstrument allows) - Production of soluble mediators can be assessed with e.g. ELISPOT, Luminex and CBA. Intracellular cytokines can be assessed with flow cytometry.
