Lecture 15 - Functional Assays Flashcards
List the issues associated with cryopreservation of cells
• 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
List 3 mitogens used for stimulating lymphocytes
- phytohaemagglutinin (PHA)
- lipopolysaccharide (LPS)
- pokeweed mitogen (PWM)
What two cell surface markers are used to determine T cell activation?
CD69 and CD40L (CD154)
Outline a basic T cell proliferation assay using thymidine
- PBMC separated from Blood
- Stimulate with mitogen (PHA or PWM) – control PBS (time dependent on mitogen)
- 3H labelled Thymidine is added (thymidine incorporated into DNA of proliferating cells)
- Wash cells to remove unincorporated labelled thymidine
- Measure radioactivity (counter)
How can flow-cytometry be used to measure cell proliferation?
- Use of fluorescent dyes to measure proliferation
- CFSE is a commonly used dye for these assays
- Can be toxic to cells in high concentration
- Must be optimised for condition as it can effect proliferation
- Cell membrane permeating dye
- Similar fluorescence as FITC
- Can be used to track cells in vivo in animal models
What is Ki-67 and how is it used to measure cell proliferation?
- Ki-67 is a nuclear protein involved in regulation of cell division
- It is expressed in cells that are actively proliferating
- Often used to detect cancer cells as they are continuously proliferating (usually with IHC)
- Recently used to measure T cell proliferation after vaccination (T cells should proliferate)
- Can be detected by flow-cytometry
• May replace thymidine method for assessing lymphocyte proliferation
respons
Outline the Chromium release assay for NK cell function.
- K562 cells are labelled with 51Cr, washed to remove excess 51Cr (K562 cells are a human chronic myelogenous leukaemia cell line that don’t express MHC I)
- Separate NK cells from blood (gradient, Dyna beads, flow-cytometry)
- Mix NK cells with labelled K562 cells, incubate 37oC (various times, ratio of cells)
- Centrifuge - Remove supernatant
- Measure/count 51Cr
- More 51Cr = more lysis of target cells
How are Treg function measured?
- Stimulate T cells with anti-CD3 and anti-CD28 antibodies
- Co-culture with Treg (7 hours)
- Measure CD69 and CD40L expression by flow cytometry
- Compare with cells not co-cultured
What is ELISPOT?
• First described in 1983
- Spleen cells from immunised mice incubated in antigen coated 96 well plates
- Cells removed from wells
- Bound antibodies (produced from cells) by ELISA
- Areas where antibodies were made formed circular zones (spots) which could
be counted
- Indicating numbers of cells being activated (to produce Abs)
• ELISPOT was latter adapted to the detection of cytokine secreting T cells
- Nitrocellulose membranes and monoclonal Abs
What are 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.
- To perform functional assays blood needs to tested no longer than 8 hours after collection
Function of 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
- Failure to express CD40L indicates that T help is impaired and downstream adaptive immune response is compromised
Function of 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
- Failure to express CD40L indicates that T help is impaired and downstream adaptive immune response is compromised
Types of antibody functional assays
- Bactericidal assays
* Opsonophagocytosis assay (OPA)
Bactericidal assays
- Patients serum mixed with known amount of bacteria (usually GNB)
- Add complement (human or rabbit)
- Plate out and count colonies to determine amount of bacteria killed (use of dyes to quantify bacteria)
Opsonophagocytosis assay (OPA)
- Killing assays involve coating bacteria (usually GPB) with Abs
- Add phagocytic cells
- Count surviving cells (plating out)
- Uptake assays involve labelling bacteria with fluorescent dye and Abs
- Add phagocytic cells
- Measure number of granulocytes with fluorescent dye by flow cytometry
