Methods

Separation of immune cells from blood

Peripheral blood mononuclear cells (PBMCs) are separated from blood using centrifugation on Ficoll-Paque. To prevent clotting, citrate is added to the blood. Ficoll-Paque is underlayed the blood in a conical tube. After being centrifuged, the following layers will be visible from top to bottom: plasma and other constituents, a layer of mono-nuclear cells called interfase (PBMC), Ficoll-Paque, and erythrocytes & granulocytes which should be present in pellet form. This separation allows easy harvest of PBMCs. Ficoll is a neutral, highly branched, high-mass, hydrophilic polysaccharide which dissolves readily in aqueous solutions. Ficoll radii range from 2-7 nm. It is prepared by reaction of the polysaccharide with epichlorohydrin.

Cell Culturing

Directly isolated cells (primary cells) or transformed cell lines are grown and maintained at an appropriate temperature and gas mixture (37°C, 5% CO2) in a cell incubator. Culture conditions vary widely for each cell type, and variation of conditions for a particular cell type can result in different phenotypes being expressed. 
Aside from temperature and gas mixture, the most commonly varied factor in culture systems is the growth medium. Recipes for growth media can vary in pH, glucose concentration, growth factors, and the presence of other nutrients. The growth factors used to supplement media are often derived from animal blood, such as calf serum, or from human serum (AB serum). Examples of primary cells used in the lab are: T cells, B cells, dendritic cells. Examples of cell lines used in the lab are: MOLT-3, SupT1, HCT-116, NIH-3T3, HEK293, U937

Viral Expansion and Viral Infection


Human herpesvirus (HHV)-6B is expanded in e.g. MOLT-3 cells and Epstein-Barr virus (EBV) in B95-8 cells. Virus is purified from the supernatant of the producer cells.
Cells are infected by co-culturing with the virus (at variable titers and time periods). Virus enter the cell following binding to its receptor (CD46 for HHV-6B and CD21 for EBV). The structural and biochemical effects that virus has on the host cell is called cytopathic effects. Virus infections may result in the death of the host cell, however, HHV-6B has evolved mechanisms to prevent the immediate cell death of the host cell and EBV is able to transform its host B cells.

Cytotoxicity

We assess cell membrane integrity for measuring cell viability or cytotoxic effects. Vital dyes, such as trypan blue or propidium iodide are normally excluded from the inside of healthy cells; however, if the cell membrane has been compromised, they freely cross the membrane and stain intracellular components. We often analyze cell viability by these methods using flow cytometry.
During T-cell cytotoxic assays, we measure the leakage from a target cell previously loaded with a substance. The substance may be a cell permeable ester that is hydrolyzed within the cell to make it impermeable. This way the substance is only detected within the supernatant if the cell membrane is compromised. Alternatively, we measure membrane integrity by monitoring the passage of substances that are normally sequestered inside cells to the outside, such as lactate dehydrogenase (LDH).

RNA Purification

RNA purification is a routine procedure to collect RNA from cells for subsequent molecular analysis e.g. PCR and cDNA synthesis. Usually an isolation kit is used.

The basic steps in the RNA extraction:
1. Lysing and homogenizing the cells with a lysis buffer to expose the RNA within and inactivating RNases.
2. Binding RNA to a silica-based membrane in a spin column by adding ethanol.
3. Washing bound RNA to get rid of contaminants.
4. Purified RNA is eluted in water.

Genomic DNA Purification

Genomic DNA purification is a routine procedure to collect DNA from blood, cells or tissue for subsequent molecular analysis e.g. PCR. Usually an isolation kit is used.
The basic steps in the DNA extraction:

1. Lysing the cells with a lysis buffer to expose the DNA within and removing proteins by adding a protease.
2. Binding nucleic acids to the glass fiber in a filter tube.
3. Washing bound nucleic acids to get rid of PCR inhibitory contaminants.
4. Washing bound nucleic acids to purify from salts, proteins and other cellular impurities.
5. Purified Nucleic Acids are recovered using an elution buffer.

Viral DNA can be purified with a similar procedure from serum, plasma, blood or cells.

Flow Cytometry

Flow cytometry is a technique for counting and examining microscopic particles, such as cells, by suspending them in a stream of fluid and passing them by an electronic detection apparatus. It allows simultaneous multiparametric analysis of the physical and/or chemical characteristics of up to thousands of particles per second.

A beam of light (usually laser light) of a single wavelength is directed onto a hydrodynamically-focused stream of fluid. A number of detectors are aimed at the point where the stream passes through the light beam: one in line with the light beam (Forward Scatter or FSC) and several perpendicular to it (Side Scatter (SSC) and one or more fluorescent detectors). Each suspended particle passing through the beam scatters the ray, and fluorescent chemicals found in the particle or attached to the particle may be excited into emitting light at a longer wavelength than the light source. This combination of scattered and fluorescent light is picked up by the detectors. By analysing fluctuations in brightness at each detector (one for each fluorescent emission peak), it is then possible to derive various types of information about the physical and chemical structure of each individual particle.

PCR and Real-Time qPCR

PCR machine The polymerase chain reaction (PCR) is a technique to amplify a single or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence. 

PCR applications employ a heat-stable DNA polymerase, such as Taq polymerase. This DNA polymerase enzymatically assembles a new DNA strand from the nucleotides, by using single-stranded DNA as a template and DNA oligonucleotides (DNA primers), which are required for initiation of DNA synthesis. Thermal cycling steps are necessary first to physically separate the two strands in a DNA double helix at a high temperature. At a lower temperature, each strand is then used as the template in DNA synthesis by the DNA polymerase to amplify the target DNA. The selectivity of PCR results from the use of primers that are complementary to the DNA region targeted for amplification.

In Real-time polymerase chain reaction, also called quantitative real time polymerase chain reaction (qPCR/qrt-PCR), is a laboratory technique used to amplify and simultaneously quantify a targeted DNA molecule. For one or more specific sequences in a DNA sample, Real Time-PCR enables both detection and quantification. The quantity can be either an absolute number of copies or a relative amount when normalized to DNA input or additional normalizing genes. 
The procedure follows the general principle of polymerase chain reaction; its key feature is that the amplified DNA is detected as the reaction progresses in real time. Two common methods for detection of products in real-time PCR are: (1) non-specific fluorescent dyes that intercalate with any double-stranded DNA, and (2) sequence-specific DNA probes consisting of oligonucleotides that are labeled with a fluorescent reporter which permits detection only after hybridization of the probe with its complementary DNA target.

Cloning of Genes

Cloning is commonly used to amplify DNA fragments containing whole genes.
The DNA to be cloned is obtained from an organism of interest and then treated with enzymes to generate smaller DNA fragments. These fragments are then combined with vector DNA to generate recombinant DNA. The recombinant DNA is then introduced into a host organism (typically an E. coli bacteria). This will generate a population of organisms in which recombinant DNA molecules are replicated along with the host DNA.

Fluorescence-Activated Cell Sorting (FACS)

Fluorescence-activated cell sorting (FACS) is a specialized type of flow cytometry. It is a method for sorting a heterogeneous mixture of cells into two or more containers, one cell at a time, based upon the specific light scattering and fluorescent characteristics of each cell. It provides fast, objective and quantitative recording of fluorescent signals from individual cells as well as physical separation of cells of particular interest.

Proliferation Assays

Proliferation of cells can be assessed by measuring the incorporation of tritiated thymidine. Cells are harvested to filter paper and counted in a scintillation counter.

Cell Cycle Assays

Cell cycle assays are performed by flow cytometry analysis of cells using dyes for DNA. Different approaches can be applied to improve the assessment of cells in the various phases of the cell cycle.

Western Blotting / Immunoblotting

A widely used analytical technique used to detect specific proteins in the given sample of tissue homogenate or extract. It uses gel electrophoresis to separate native or denatured proteins by the length of the polypeptide (denaturing conditions) or by the 3-D structure of the protein (native/ non-denaturing conditions). The proteins are then transferred to a membrane (typically nitrocellulose), where they are detected using antibodies specific to the target protein.

ELISA

ELISA, also known as an enzyme immunoassay (EIA), is a technique used to detect the presence of an antibody or an antigen in a sample. In simple terms, in ELISA, an unknown amount of antigen is affixed to a surface, and then a specific antibody is applied over the surface so that it can bind to the antigen. This antibody is linked to an enzyme, and in the final step a substance is added that the enzyme can convert to some detectable signal, most commonly a color change in a chemical substrate

Transfection

Transfection is the process of deliberately introducing foreign DNA into eukaryotic cells. Various methods exist, e.g.:

In transient transfection the DNA introduced in the transfection process is usually not integrated into the nuclear genome, and the foreign DNA will be diluted through mitosis or degraded.

In stable transfection the transfected gene actually remains in the genome of the cell and its daughter cells. To accomplish this, a marker gene is co-transfected, which gives the cell some selectable advantage, such as resistance towards a certain toxin. Some (very few) of the transfected cells will, by chance, have integrated the foreign genetic material into their genome. If the toxin is then added to the cell culture, only those few cells with the marker gene integrated into their genomes will be able to proliferate, while other cells will die. After applying this selective stress (selection pressure) for some time, only the cells with a stable transfection remain and can be cultivated further.

Electroporation is a transfection method where micro-sized holes are created transiently in the plasma membrane of cells under an electric discharge, allowing the DNA to enter into the cells.

cDNA Synthesis

Complementary DNA (cDNA) is DNA synthesized from a mature mRNA template in a reaction catalyzed by the enzyme reverse transcriptase and the enzyme DNA polymerase. Reverse transcriptase operates on a single strand of mRNA, generating its complementary DNA based on the pairing of RNA base pairs (A, U, G and C) to their DNA complements (T, A, C and G respectively).
cDNA is often used to clone eukaryotic genes in prokaryotes and can be used to express a specific protein in a cell that does not normally express that protein by transferring the cDNA that codes for the protein to the recipient cell.

Confocal Laser Scanning Microscopy

Confocal Laser Scanning Microscopy (CLSM or LSCM) is a technique for obtaining high-resolution optical images with depth selectivity. The key feature of confocal microscopy is its ability to acquire in-focus images from selected depths, a process known as optical sectioning. Images are acquired point-by-point and reconstructed with a computer, allowing three-dimensional reconstructions of topologically complex objects.