Table of Contents
UNIT 1 LECTURE ... 2
DESCRIBE THE USE OF FLUOROCHROMES TO ANALYSE CELLULAR PROLIFERATION AND CELL TRACKING ...2
MEASURING ANTIGEN-SPECIFIC T CELL RESPONSES BY FLOW CYTOMETRY ...2
TRANSGENIC MICE ...3
WHAT IS FLOW CYTOMETRY? ...6
COMPONENTS OF A FLOW CYTOMETER ...6
FORWARD SCATTER (FSC) AND SIDE SCATTER (SSC) ...6
HOW DO WE USE FLUOROCHROMES (AKA FLUOROPHORE) ...7
LYMPHOCYTE POPULATION ...7
GATING STRATEGY FOR FACS ANALYSIS ...8
**CONTROLS FOR FACS ...8
T CELL DEVELOPMENT ...9
TCR REARRANGEMENT ...9
T CELL SELECTION PROCESS ...9
IMMUNOHISTOCHEMISTRY... 10
STRUCTURE OF THE SPLEEN AND THYMUS ... 11
IMMUNOHISTOCHEMISTRY VS FLOW CYTOMETRY ... 12
UNIT 2 ... 13
PATTERN RECOGNITION RECEPTORS OF THE INNATE IMMUNE SYSTEM ... 13
DC ACTIVATION & MATURATION ... 13
DC STIMULATION OF T CELLS... 14
ELISA... 15
ELISPOT ASSAY USED TO DETECT AND COUNT THE CYTOKINE PRODUCING CELLS ... 16
FLOW CYTOMETRY - DETECTION OF NUMBER AND TYPE OF CYTOKINE SECRETING CELLS ... 17
REAL TIME RT-QPCR ... 17
SYBR GREEN QPCR ... 17
HAEMOCYTOMETER ... 19
UNIT 3 ... 21
PRINCIPLES OF T CELL RECEPTOR RECOGNITION OF MHC/ANTIGENS ... 21
DEVELOPMENT OF T CELLS AND NKT CELLS ... 21
WHAT IS T CELL BIAS?... 22
FACTORS OF A TCR ARE IMPORTANT FOR RECOGNITION OF MHC AND CD1D ... 23
PRAC 1C
Immunohistochemistry
Immuno = antibodies, histo = tissues, chemistry = biochemical reactions (rxn that causes colour change)
It is the application of monoclonal and polyclonal antibodies to detect specific antigens in tissue sections
Use of various enzyme labels (e.g., peroxidase, alkaline phosphatase) on the antibody Visualise antibody binding with substrate (insoluble) for the enzyme
o This leads to localised deposit of colour reaction (chromogen) Light microscope for observation
Can also use fluorescent label for fluorescent immunohistochemistry Mechanisms of Action:
A primary antibody against the antigen is added. Then the primary Ab will be bound to the Ag. Excess Ab is washed off.
A secondary antibody is added that has specificity towards the Fc region of the primary antibody. The secondary antibody is conjugated with Horse radish peroxidase (HRP) polymer.
Excess Secondary Ab that is not bound is washed off.
Then add substrate for HRP which is DAB (a chromogen). This will stain the cells with the Ag brown which is visualised under the microscope.
Tissue preparation
We have to preserve antigens/epitopes of interest and preserve tissue morphology Two common techniques used:
o Fix tissue with formalin, then embed in paraffin (Paraffin Embedded Tissue) o Embed tissues in cryoprotective medium and then freeze tissue sample at -80C.
Both techniques need microtome to prepare 4-10µm thin sections of the paraffin block containing the tissue/frozen tissue
Transfer section to glass slide, use for immunohistochemistry Paraffin embedded tissue Frozen Tissues Advantages:
Can be stored at room temperature (do not need specialised equipment for storage)
Formalin and paraffin embedded tissues crosslinks the proteins and maintains tissue structure (preserves cell morphology
Advantages:
Quick
Preserve enzyme activity for enzyme histochemistry and antigenicity Allow molecular genetic analysis Proteins are still in their native state Good antigen preservation
Disadvantages:
Time-consuming Formalin is toxic
The samples is no longer biologically active (enzyme activity not preserved) may affect antigens
Disadvantages:
Unfixed tissue must be fixed before staining
Rapid deterioration of samples once it is in room temperature
Storage equipment is expensive
*Controls for immunohistochemistry:
Use isotype control staining (negative control) to ensure there is no non-specific binding same Ig class, same enzyme label but different specificity
Positive control to avoid any false negative results Detection Systems Chromogens
Various chromogens (substrates) for HRP:
o DAB brown precipitate o AEC red precipitate
o Vina Green green precipitate Can have different staining patterns:
o nuclear staining, membranous staining, cytoplasmic staining and connective tissue staining
Counterstain: to visualize histological features of the tissues/cells
o Use hematoxylin: stains cellular cytoplasm pale blue and nuclei dark blue o or eosin: stains cytoplasm light pink
To prepare for long term storage: add coverslip, seal sample for protection using purpose made mounting media.
Structure of the spleen and thymus
Spleen
Important in the filtration of blood and immune function Lymphocytes enter and leave the spleen via blood vessels Red pulp is the site of RBC disposal
The lymphocytes that surround the arteriole running through the spleen, forms white pulp.
The sheath of lymphocytes surrounding the arteriole is called periarteriolar lymphoid sheath (PALS) and mainly consists of T cells
Lymphoid follicles that occur at intervals along it consists mainly of B cells
Marginal zone surrounding the follicles is rich in macrophages and marginal zone B cells which can quickly differentiate into plasma cells
Thymus
Consist of cortical (outer) regions and medullary (central) regions
The thymocytes in the outer cortical cell layer are proliferating immature cells, whereas the deeper cortical thymocytes are mainly immature T cells undergoing thymic selection.
Medulla consists of mature thymocytes (medulla = SP T cells, cortex = DN & DP T cells) Cortical epithelial cells (aka stromal cells) assists in positive selection
Medullary epithelial cells expresses self-Ag (AIRE)
DC & macrophages present Ag to T cells for negative selection Ha al co p cle a e i e of cell deg ada ion
We can see that there is more cells in the cortex (DN & DP), and lesser in the medulla. This is beca e majo i of h moc e don i e po i i e and nega i e elec ion The efo e these are degraded.
Experimental Design Q&A
Why is it important to not let slides dry out
o Tissue integrity will be loss and the probability of non-specific binding will increase What is the purpose of blocking step
o To prevent non-specific binding of Ab to the glass slides and the tissues Why do we need to use isotype control Ab?
o To validate the results by checking to see if he e an non-specific binding What solution would you dilute the Abs in?
o PBS + a bit of FCS
The purpose of washing step is to wash away excess Ab
Sections were stained with haematoxylin to visualise the tissue morphology as it stains the cytoplasm pale blue and the nucleus dark blue
Immunohistochemistry vs Flow cytometry
Immunohistochemistry Flow cytometry
A qualitative approach A quantitative approach
Cannot quantify the number of cells Can quantify the number of cells that are expressing the cell surface marker Allows the analysis of tissue morphology
(phenotypic)
Cannot analyse tissue morphology as single cell suspensions are made
No functional analysis can be made unless we use the frozen tissue sections and perform enzyme bioassays on it
Can analyse the cell surface marker expression/
look at cell proliferation, TF expression and cytokines production
Only needs a light microscope Needs a flow cytometer
More Time consuming Less time consuming and can look at large number of cells in one time
For tissue sections that are fixed -> the cells cannot be used for further analysis
Cells are still viable for further analysis Allows binding to intracellular proteins/antigen
(e.g., CD3 is present intracellularly for RAG-ko mice, but not on the surface -> can still be stained)
binds to the cell surface marker only (unless fix and permeabilise the cell)
