W11L15 - Functional Assays Flashcards
Functional Assays
Can measure amounts of proteins - antibodies - complement levels Cellular assays - can measure numbers of cells - can look at morphology of cells Functional assays determine cellular effectiveness
Cryopreservation of Cells - Overview
To perform functional assays blood needs to tested no longer than 8 hours after collection
- peripheral blood mononuclear cells (PBMC) start to lose functionality
However, it is not always possible to have fresh blood
Utility of cryopreservation of cells is widely recognised
Cryopreservation of Cells - Goals and Issues
Goal is to be able to freeze and thaw cells without compromising cell viability or functionality
- process cells within 8 hours of collection
Main issue with cryopreservation is the formation of water crystals
- causes major disruption mainly to cell membranes
-cells rupture on thawing
Dehydration of cells while freezing
- shrinkage of cells
- loss of function
Prevent osmotic trauma
- cells burst on thawing
Cryopreservation of Cells - Agents Used to Improve
Agents used while freezing to avoid water crystals forming
- glycerol
-dimethyl sulfoxide (DMSO) - for PBMC this agent is mainly used
To prevent dehydration
- inhibit cell metabolism while freezing
- rate controlled freezing (step wise reduction in temperature)
Maintain osmotic pressure
- use of serum in media while thawing
Protocol for Cryopreservation of PBMC
Ficoll-Hypaque separation of PBMC
Wash cells in cold PBS (4°C)
Resuspend cells in cold (4°C) FCS containing 10% DMSO (on ice)
- cells need to be counted and suspended in FCS/DMSO at approximately 10^7 PBMC/ml
Cells aliquoted into cryo-vials (2ml per vial)
- kept on ice
Cool cells at approximately –1°/minute (24hours)
- Nalgene, BioCision, cryomed freezing apparatus
- alternatively place at -20°C for 1-2 hours
Place into -80°C storage
- cells remain functional for approximately one week
Then place into LN2
(-150°C) for long time storage
- cells remain viable and functional for decades
Thawing Cells
Cryovial removed from -80°C or LN2
Incubated at 37°C (water bath) with agitation
When content of vial almost all fluid transfer to 15 ml polypropylene conical tube
Resuspend in RPMI media with 10% FCS (or human AB serum)
- media is added slowly (1 ml/minute) with mixing - 5 mls
- add extra 5-10 mls
- anti-clumping agents can be used in media (Dnases - Benzonase)
Cells washed (PBS or media) x2 by centrifugation
Resuspended, counted and adjust for concentration, test for viability
Cell Viability
Many assay available
Trypan blue staining - simple and quick
- mix a drop of cell suspension with 5-10 μl of trypan blue
- load into a haemocytometer (cell count)
- non liable cells are permeable to trypan blue - stained blue
- liable cells impermeable to trypan blue - unstained and retractile
Calculate % of viable cells
Total number of viable cells (haemocytometer)
Fresh vs Cryopreserved Cells
Mitogen induced T cell proliferation assays
- high correlation between fresh and cryopreserved cells
- slight decrease in proliferation with cryopreserved cells
Cytotoxic assays
- cytotoxic T cells (CD8+) comparable
- NK cells have reduced lysis of target cells after cryopreservation
Cytokine assays
- INF-ɣ production same
- IL-2 comparable
Surface markers (flow cytometry)
- CD4 and CD8 consistent results (even after decades of storage for cryopreserved cells)
- other markers also consistent
- “must gate on only viable cells”
B cells
- memory B cells survive cryopreservation well
- plasmablasts do not survive cryopreservation - care for any assay involving antibody production
Lymphocyte Activation
Assays to determine lymphocyte ability to activate after stimulation
Important for several immune diseases
- primary deficiency (genetic)
- altered due to therapy
Two approaches:
1. Measurement of surface or intracellular markers (immunophenotyping) after stimulation
2. Direct measurement of function after stimulation - proliferation, activation or cytotoxicity
Mostly done on T cells (but also B cells and NK)
T Cell Activation and Function
Naïve T cells recognise antigens and become activated
- this results in expansion of the antigen specific T cells which then differentiate into effector and memory T cells
- happens in secondary lymphoid organs (LN) and require presentation by APC
In vitro T cells can be activated by certain stimulants
- mitogens (substances that stimulate cell division)
Response to Mitogens
Mitogens are very potent stimulators of T cell activation
Independent of antigen specificity
- some T cells can respond to mitogens even if they cannot respond to antigen
- T cell response to mitogen is considered less sensitive but more specific test for aberrant T cell function
Mitogen stimulation increases intracellular Ca2+ essential for proliferation
Different mitogens stimulate differently
- PHA is a strong T cell mitogen but does not stimulate B cells
- PWM is a weak T cell mitogen but also stimulates B cells
Non-mitogen stimulation
- anti-CD3 antibody with IL-2
- antigen response (more sensitive but less specific)
Markers of T Cell Activation
After stimulation with a mitogen it is possible to detect cells markers by flow cytometry
- specific labelled antibodies to markers
The two most common markers used are CD69 and CD40L (CD154)
Markers of T Cell Activation - CD69
Signal transmembrane glycoprotein involved in T cell proliferation
Expressed at very low levels on resting T cells
Rapidly upregulated after TCR stimulation
Within 1 hr with peak expression 16 -24 hrs (undetectable after 72 hrs)
Different mitogen stimulation
- PHA induced after 24hrs and past 72 hrs
- Phorbol myristate acetate (PMA) and ionomycin induce CD69 after 2 hrs
Inability to express CD69 after stimulation indicates impaired T cell function
Markers of T Cell Activation - CD40L (CD154)
Binds to CD40 on APC
Activates several intracellular pathways
Expression is induced in 1-2 hrs after TCR stimulation
Peak expression at 6 hrs, declines after 16-24 hrs
However, in combination with anti-CD28 or IL-2 co-stimulation CD40L expressed for several days
Expression on CD8 cells is weak and only little increase after mitogen stimulated
Expression on resting CD4 cells is weak but is greatly increased by mitogen stimulated
Test for CD4 activation - function of T help
Failure to express CD40L indicates that T help is impaired and downstream adaptive immune response is compromised
Assessing CD40L Function
Assessed by its ability to bind to soluble CD40-muIg
(CD40 fused to Fc portion of mouse IgG)
- cells mixed with labelled CD40-muIg
- labelled anti-mouse IgG Fc
- if binds to CD40L can detect by flow cytometry
- failure to bind is associated with X-linked hyper-IgM syndrome
