Flow cytometry Flashcards
how can cells of the immune system be identified
- immune system cells express surface molecules
- these can be used to classify and identify cells - O THEY ARE MARKERS
- can be both external (common) or internal
how did CD method of classification come about
- CD is cluster of differentiation
- not all markers/CD molecules are SPECIFIC for 1 cell type (some are rexpressed by a number of differento cells)
what do T cells express to identify them
- CD3
no CD19,21 OR 14
what do B cells express to identify them
- CD19 and CD21
no CD3 or 14
what do monocytes express to identify them
- CD14
no CD3, 10 or 21
how do we detect the presence of these markers
using antibodies
what do naive B cells express
what do they secrete when they encounter antigen
NAIVE:
- express antibodies/membrane immunoglobulin (IgM and IgD) as part of BCR
- when they encounter antigen they SECRETE IgM antibodies
- later in the immune response the same variable region may be expressed as IgA, IgG or IgE antibody classes
what is the role of variable regions in B cells
antigen binding sites
what is the role of constant regions in B cells
effector function (binding FcRs, activation of complement)
what are the subtypes of of IgG
can be further divided into subclasses e.g. IgG 1, 2a, 2b & 3 in mice and IgG1, 2, 3 & 4 in humans which are also called ‘isotypes’
- structures vary slightly slighty
what can antibodies be used on
- cells (single cells, mixtures, tissues)
- secreted/ biological products (cytokines, hormones, growth factors) (
how are commercial antibodies prepared
- from a specific B cell (monoclonal antibody: MAb) or from a pool of B cells specific for different epitopes of the same antigen
- can be tagges with FLUORESCENT MARKERS, ENZYMES or RADIOACTIVE ISOTOPES
what can antibody detection of cell surface or internal molecules be used for
- identify/count cells
- purify cells/tissues/secreted products
- examine cellular activation/differentiation status
- examine pathology, expression of abnormalities
which techniques use antibodies
- Affinity purification
- Immunoprecipitation
- ELISPOT
- Immunofluorescence
- Histology (immunohistochemistry)
- ELISA
- Flow cytometry
describe ELISA
(Enzyme-Linked ImmunoSorbant Assay)
- Detection of secreted soluble products such as cytokines +/- an amplification step with biotin/streptavidin/enzyme linked antibodies
describe flow cytometry
Uses the light scattering properties of single cells in suspension in conjunction with laser excited fluorescence from surface marker specific Antibodies with fluorescent tags (fluorophores) to detect expression of surface or internal markers and or cellular products
what are the key features of flow cytometry
Hydrodynamic or acoustic focussing of a single cell suspension•Cell size and granularity are detected by light scattering patterns
- Cells can be ‘stained’ or ‘labelled’ for a specific marker with fluorescently tagged antibodies or dyes (fluorophores)
- Fluorophores are excited by a laser, and emit fluorescence at a longer wavelength
- Fluorescent emissions are filtered, quantified and displayed/stored by the software
- Multiple, simultaneous measurements on the same cell can be made
- Large numbers of cells can be accurately measured very quickly – also allows for the measurement of rare cell type
what is immunophenotyping using flow cytometry
- Abs specific for cell surface markers on immune cells (CD molecules) can help identify/count/analyse cells
- By using fluorophores with different excitation/emission profiles, the researcher can MULTIPLEX – use combinations of fluorescently labelled antibodies/dyes to measure multiple cell parameters e.g. Ab to marker A-FITC and Ab to marker B-RPE in one tube, allowing the researcher to discriminate between different cell types in the same sample
- new fluorophores are continuously being discovered and with increased numbers of lasers, most machines can currently measure up to 28 colours, in the near future this will be up to 50 – this will lead to very complicated analysis
what is FITC
- fluorophore
- fluorescein isothiocyanate
- excitation laser is at 488 nm
- emission detector is at FL-1 green
what is PE
- fluorophore
- R-phycoerythrin
- excitation laser is at 488nm
- emission detector is FL-2 yellow
what is APC
- allophycocyanin
- excitation laser is at 633nm
- emission detector is FL-4 red
what is the routine application of flow cytometry
- CD4+/CD8+ balance in HIV infected patient
what are the research applications of flow cytometry
- Live/dead counts•Ploidy studies
- Cell cycle analysis
- Ca++ flux & morphology changes (drug treatment effects)
- Morphology changes (drug treatment effects)
- Cell activation
- Cytokine production
- Cell subtype composition (T reg, gdT cell, CD5+ B cell populations)
- Bacterial infection (Green Fluorescent Protein expressing bacteria)
- Cell sorting
how can the no of T helper cells be calculated
what is the cellular composition of whole blood
WHOLE BLOOD: (step 1)
- Monocytes ~3-5%, lymphocytes ~20-30%, neutrophils ~70%, NK cells (large granular lymphocytes), eosinophils ~1% of the leukocytes (white blood cells)….others
- RBC and platelets can cause problems as they can create a lot of noise
- O we add a LYSIS step to remove RBCs and platelets
step 2
IDENTIFY LYMPHOCYTES from other leukocytes
- lymphocytes are small and not very granular – so can use forward and side scatter properties (FSC and SSC)to distinguish them from the other leukocytes (WBCs)
step 3
USE FLUORESCENT Abs to identify lymphoctes
- ALL T cells express CD3 but HELPER T CELLS SPECIFICALLY express CD4
- Mouse anti-human CD3 antibody is conjugated to FITC fluorophore. Binds to CD3 molecules on the cell surface CD3-positive cells fluoresce when pass through the laser detect green fluorescence
- Mouse anti-human CD4 antibody is conjugated to PE fluorophoreBinds to CD4 molecules on the cell surfaceCD4-positive cells fluoresce when pass through the laser detect yellow-red fluorescence
how can we remove RBCs and platelets from blood sample
incubate blood in low conc of AMMONIUM CHLORIDE in water before running on flow cytometer
what kind of scales are side scatter (SSC) and forward scatter (FSC) measured on
linear scales
- size on x axis and granularity on y axis
when using fluorescent Abs how will th edifferent cells react
T HELPER CELLS
- express CD4 O will bind one set of Abs and will express with R-phycoerythrin (PE) and fluoresce RED
- express CD3 O will bind another set of Abs and fluoresce GREEN
CYTOTOXIC T CELLS
- express CD3 O will bind one set of Abs and fluoresce GREEN
- express CD8 O cannot bind the CD4 specific Ab O does not fluoresce RED
B CELLS AND MONOCYTES
- do not express (usually) CD3 or CD4 O do not fluoresce
in the results of lysed whole blood fluorescent dot plot what can be seen
- bottom left quadrant:
CD4 and CD3 NEGATIVE cells (B cells, NK cells, Monocytes, Granulocyte) - top left quadrant:
CD4 POSITIVE and CD3 NEGATIVE cells (monocytes and some DCs) - bottom right quadrant:
CD3 POSITIVE and CD4 NEGATIVE cells (T cytotoxic cells(and some γδT cells, NKT cells) - top right quadrant:
CD4 POSITIVE AND CD3 POSITIVE cells (T HELPER CELLS)
how can we make the fluorescent dot plot easier to read
- we can COMBINE the FSC and SCC (forward and side scatter) data with the fluorescent data and draw A GATE that displays only cells with certain characteristics
what controls are used in flow cytometry
1) cells only control
2) isotype controls
describe cells only control in flow cytometry
- Unstained cells (no Abs/fluorophore) provide information about the background ‘autofluorescence’ of each cell type
- The operator adjusts the Photomultiplier Tube (PMT) detectors so that the cells are situated in the bottom left of the quadrant
describe isotype controls in flow cytometry
- Antibodies can bind to Fc Receptors
- Species differences (mouse Ab/IgG and human FcRs) should reduce this
- Antibodies can stick non-specifically to the cell surface
- The subclass or ‘isotype’ of the antibody affects non-specific binding
O IT IS IMPORTANT TO HAVE A TUBE CONTAINING ISOTYPE CONTROL REAGENTS
- An antibody of the same: ‘isotype’, fluorophore, and amountBut it recognises an irrelevant antigen eg KLH not present on mamalian cells. This way we can discriminate ‘specific’ antibody binding from ‘non-specific’ background binding to Fc Receptors or to the cells. It allows the researcher to put the quadrant in the correct place to determine positive events
describe the test sample in flow cytometry
- The test sample will have fluorophore tagged antibodies specific to the markers added which will MAINLY bind specifically (via the Fab regions) as long as the marker is present on the cells and some non specifically (eg by Fc regions binding to Fc receptors, or to any region sticking to a cell)
- By comparing the isotype control to the test sample, we are able to determine the events that are positive, and set the quadrantsThe statistics of these quadrants can then be recorded – the number of cells counted, the % cells in each quadrant and also the mean fluorescence intensity of the bound antibodies
hiw us tge proportion of T helper cells calculated
1) calculate which % of lymphocytes are T cells:
Proportion of lymphocytes that are CD3+ = 35+25=60%
2) which % of lymphocytes are T helper cells
(% of lymphocytes that are CD3+ and CD4+)
3) which % of T cells are T helper cells?
(proportion of CD3+ cells that are CD4+) ie
( % CD3+ CD4+) /
(% CD3+ CD4+ ) + (% CD3+ CD4-) x 100
