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
