Cell stimulations

Throughout this study all cell stimulations were carried out in their respective mediums (Section 2.2) and incubated at 37ºC in a 5% CO2 humidified atmosphere. 1,000,000 cells per sample were stimulated during the course of this study. Ligand concentrations were kept constant throughout this study (Table 2.3).

Ligand Concentration Lipopolysaccharide (LPS) 100ng/ml V antigen 50μg/ml V1 50μg/ml V3 50μg/ml V4 50μg/ml V5 50μg/ml

Table 2.3: Ligand concentrations used in this study. All concentrations were kept constant throughout.

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2.6.1. 25cm

flask stimulation

Human monocytic leukemia (THP-1) cells were cultured in 80cm2 flasks (Nunc) and stimulated in flasks with a surface area of 25cm2 (Nunc). Sterile conditions were practiced throughout. THP-1 cells were counted using a hemocytometer (Section 2.3). The cell to volume ratio was rectified to 1,000,000 cells/ml. The cells were then stimulated.

Mouse leukaemic monocyte macrophage (RAW 264.7) cells were stimulated in flasks with a surface area of 25cm2 (Nunc). Sterile conditions were practiced throughout. The growth medium was aspirated off and the cells were washed with 3ml of fresh medium. 2ml of medium was then added to the flask. The cells were then stimulated.

2.6.2. Lab-Tek

slide stimulation

For confocal microscopy mouse leukaemic monocyte macrophage (RAW 264.7) cells were grown and stimulated on 8 well glass slides (Nunc Lab-TekTM Chamber SlideTM System). The growth medium was aspirated off and the cells were washed with 400μl of fresh medium. 150μl of growth medium was then added to each well. The cells were then stimulated.

2.7. Immunofluorescence

Immunofluorescence is a technique whereby specific molecules are labelled and visualised with fluorescent antibodies. Immunofluorescent labelling can be utilised to find the relative abundance and localisation of a chosen antigen. Fluorochromes that emit light of a set wavelength are conjugated to antibodies. Fluorescence is emitted from fluorochromes that become excited when exposed to a laser beam which results on the emission of light at a certain wavelength, which in turn can be imaged and/or quantified by a number of techniques. The development of fluorescent labelling as a research tool has proven invaluable.

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Fluorescence is emitted from fluorochromes that become excited when exposed to a laser beam of the correct wavelength. Electrons in the fluorochrome move into a higher energy state when a photon from the laser is absorbed. The atoms are said to change from their “ground state” to an “excited state”. The emitted light is detected and can be used to quantify and determine the localisation of a chosen antigen.

There are a number of different fluorochromes available which have different excitation and emission wavelengths. Such availability of fluorochromes coupled with high specificity antibodies allows multiple labelling of molecules and/or structures in a single sample at any one time. The table below shows the different fluorochromes used throughout this study (Table 2.4).

Fluorochrome Excitation λ Emission λ Colour

Fluorescein isothiocyanate (FITC) 495nm 519nm Green

Phycoerythrin (PE) 547nm 572nm Yellow

Cy3 553nm 556nm Red

Cy5 650nm 667nm Blue

Alexa fluor 633 632nm 647nm Red

Table 2.4: Fluorochrome excitation, emission and observed colour of fluorochromes used in this study.

Immunofluorescence can be carried out through two main tests – direct and indirect.

2.7.1. Direct immunofluorescence

Direct immunofluorescence involves the direct binding of a fluorochrome conjugated primary antibody/ligand to the antigen of interest (Figure 2.6). A specific antigen will be detected by the antibody, and the fluorophore it is attached to can be subsequently detected via microscopy or flow cytometry.

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Figure 2.6: Direct immunofluorescence. Direct binding of fluorescein isothiocyanate (FITC) fluorochrome conjugated antibody to a target antigen. On excitation the labelled antibody emits fluorescence that can be detected and quantified.

When using direct immunofluorescence to label a protein in low abundance a weak signal may be achieved. To surpass this indirect immunofluorescence labelling can be used.

2.7.2. Indirect immunofluorescence

Indirect immunofluorescence differs from direct immunofluorescence in that it involves two compatible antibodies: an unlabelled primary antibody specific for the antigen of interest; and a fluorescently conjugated secondary antibody specific for the primary antibody (Figure 2.7). The structure of an antibody makes this possible, as it has two regions: a Fc region (fragment crystallisable region) and a Fab region (fragment antigen-binding region). The Fab region contains variable sections that determine which antigen is bound, whilst the Fc region is constant in a class of the same species. Antibodies can be designed in such a way that they contain the same Fc region but different Fab regions. In this way, primary antibodies with the same Fc region can be used to detect various antigens (due to differing Fab regions), and still be detected by a

Antigen FITC labelled

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single fluorescently-conjugated secondary antibody specific for the Fc region of the primary antibody.

Figure 2.7: Indirect Immunofluorescence. Indirect immunofluorescence involves the binding of an unlabelled primary antibody to a chosen receptor, then the further binding of a fluorescein isothiocyanate (FITC) conjugated secondary antibody to the primary antibody. On excitation the labelled secondary antibody emits fluorescence that can be detected and quantified.

The primary and secondary antibody binding is species-specific. If a goat TLR4 primary antibody was used for example, an anti-goat fluorochrome conjugated secondary antibody will have to be used with this, such as a rabbit anti-goat antibody.

Indirect immunofluorescence has the advantage of being more sensitive than direct immunofluorescence because more than one secondary antibody can bind to any one primary antibody, thus giving a stronger signal.

Unlabelled primary antibody FITC labelled secondary antibody Antigen

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