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    Friday, 14 March 2014 13:18

    What cells of the immune system exist?

    B-targeting-rosa26-feeder-cells-human-primary-cells-mefCells of the immune system are responsible for the maintenance of the immune reactivity of the organism against penetrating viral and bacterial pathogens in the human body, which sometimes can cause dangerous health infections. Initially, the circuit is driven by the production of pluripotent stem cells that differentiate and give rise to clones of B-and T-cell lymphocytes, neutrophils, eosinophils, basophils, monocytes, dendritic cells, mast cells and red blood cells.

    Neutrophils are a special type of immune system cells, which are differentiated from myeloid stem cells. They migrate into the bloodstream at himiotaksichen signal from the body. By way of specific Fc-receptor fixed opsonized by the complement system and pathogen invasion within particles. They have the ability to destroy pathogens directly emptied by the strong bactericide them.

    Eosinophils are another type of special cells of the immune system. These granules have a strong cytotoxicity to the endothelium, epidermis, neuronal cells and proteins. Their main function is to synthesize mediators and cytokines.

    Basophil cells and mast cells contain receptors for immunoglobulin E. Their main function is to synthesize prostaglandins Class 2. They also provide protection against parasites come against immunoglobulin E-mediated allergic reactions. In this way, the body remains secure during falling of animal parasites in the food, which is particularly common in the consumption of food or poorly cleaned poorly processed meat products.

    Monocytes have a cytoplasm filled with specific granules. They originate from myeloid stem cells and differentiate into macrophages. Their main function is to phagocytose emptied environmental pathogenic microorganisms. These processes are carried out after activation by interferon or TNF, macrophages subsequently acquire strong cytotoxic activity and have a strong bactericidal effect against pathogens become trapped in the body.

    Dendritic cells are antigen presenting cells. They can migrate into the blood and lymph. They are activated by irritation of those who fell in the human pathogenic microorganisms. The next stage of their operation is not present on its surface MHC molecules of the system that is the major histocompatibility complex on. Also involved in the activation of clusters of differentiation in the body - CD-cells.

    T lymphocytes very well may be defined as "the heads of special operations to eliminate the invasion and destruction of pathogenic microorganisms." They are produced in the bone marrow pluripotent stem cells and then migrate to the thymus and mature there. Subsequently differentiate and acquire the specific T-cell reactivity - TCR.

    Accordingly, this type of cells are involved in the differentiation to a CD- cells , which are clusters of differentiation. CD4 + T - cell specific agents have adjuvant - inducing function . They give rise to a whole new spot in the production of protective cells against emptied into a person's body pathogenic microorganisms. CD8 + T-cell lymphocytes are the next most important clusters that need to be addressed. They have strong cytotoxic and suppressor role. Cast directly to the differentiation of killer cells and suppressor cells when the body is facing the threat of invasive blood-borne viruses and bacteria.

    The role of the immune system is to protect the body against foreign antigens, and do not react to cause outbreaks of disease events in the human body. They also prevent the occurrence of cross-reactions between antigens and the invasion of the body's own antigens. That function of the immune system is performed by the T-cells that acquire the ability to execute it in the thymus after three-step reactions.

    - The first step is differentiation of a double negative T cell lymphocytes. These are CD4-and CD8-cells.
    - The second stage is the occurrence of double positive cells - to develop CD4 + and CD8 + cells.
    - The third stage is the mixed cells, where they meet a single positive and single negative T-lymphocytes. There are two possible combinations. The first one is the manifestation of CD4-and CD8 + cells, and the second expression of CD4 + and CD8-cells.

    B-cell lymphocytes are another important cellular arm of the immune reactivity of the organism against environmental emptied the invasion of microorganisms. They are involved in humoral immunity by synthesis of antibodies. Each mature B lymphocyte produces only one type specific antibody that is expressed on the surface, such as a B cell receptor. Antibodies react against the onset of the invasion antigens.

    NK-cells are a specific cell type. They can be defined as "a SWAT team to fight pathogens." Their name comes from the abbreviation natural killers, which means that they kill directly taken into the body microorganisms without subjecting them to any attempts to phagocytic or neutralization. NK-cells are most pronounced cytotoxicity compared with all other members of the immune response. Thus limiting the spread of virus-infected cells in the human body.

    Dr. Ivan Campeotto

    MRC Centre for Molecular Bacteriology and Infection
    Imperial College London
    United Kingdom

    In my current project at Imperial College London, I am focusing on several proteins from different human pathogens. One of these proteins forms crystals, which tend to be looped with the unique c axis of ~500Å in length and oriented in parallel with the X–ray beam. This led to a severe spot–overlapping problem, which was resolved with the aid of a kappa goniometer.
    Gentaur products came to the rescue in different steps of this project.
    The first issue to solve was the fact that crystals grew on layers of microcrystals mixed with protein skin, which, when disturbed with a traditional loop, would break into many pieces, enter the loop and thereby abolish diffraction. The Microloops™ allowed for precise selection of the desired crystals without perturbation of the drop, so that skin-free crystals inside the loop could be obtained.

    targeting-rosa26-feeder-cells-human-primary-cells-mef-1

    Figure 1. Micro–crystals were used for in situ at Diamond beamline i04–1. X–ray beam and ruler are represented in red.Another problem was the reproducibility of the crystals, when crystallization trials were set–up in 96, 48 or 24 well plate formats.  I decided to try the new in–situ plates and to compare them with traditional 96 well, 48 well and 24 well plates. This experiment was performed on the same day and using the same protein batch. Whilst the other plates gave either only micro–crystals (<1μm) or failed completely to produce crystals within a given crystallization condition range, the Gentaur plates gave crystals of about 30μm in size (Fig.1)!

    targeting-rosa26-feeder-cells-human-primary-cells-mef-2

    Figure 2. In situ diffraction at the Diamond Light Source synchrotron from Gentaur plate (beam line i04–1).In addition the Gentaur in situ plate was easily transported to the synchrotron (Diamond, beamline i04–1), as it could be packed vertically. Diffraction experiments were performed in situ and crystals diffracted approximately to 3.8Å (Fig.2) with a very low background diffraction compared to competitors.
    In my opinion the greatest benefits were: crystal reproducibility, crystal–friendly plate transport and low background scattering. There are a few others benefits, which are not less important, such as the possibility of shooting the crystals with better orientations for data collection or the possibility of testing lattice disorders from data collected at RT vs. cryo data. These are all applications, which I am currently exploring at the i24 beamline at the Diamond Light Source (Oxford, UK), where complete data collection is currently achievable in situ.
    We all know that obtaining crystals and manipulating crystals represent a continuous challenge; therefore I would like to thank Gentaur for producing innovative products, which assist us crystallographers not only in delivering better results but also in overcoming experimental obstacles, which otherwise could severely delay or hamper our work.

    Gentaur’s In Situ 1 crystallization plates

    targeting-rosa26-feeder-cells-human-primary-cells-mef-3 targeting-rosa26-feeder-cells-human-primary-cells-mef-4

    dr4-stem-cell-differentiation-neural-stem-cells

    Handling and mounting tiny crystals is challenging

    Small crystals dehydrate quickly and are difficult to mount

    Small crystals (<100 μm) seem to swim away from a mounting tool. If you do manage to mount them, they end up on the neck instead of in the aperture, and by the time you’re ready to flash cool, the crystal and/or your drop have dried out.



    Analysing the problem

    Stoke’s law and laminar flow

    Mounts seem to push small crystals away, as if the mount were too hydrophobic

    What you see is a simple demonstration of laminar flow and Stoke’s law. When you move the mount through a crystal-containing drop, the liquid flows laminarly around it. If the crystal’s density matched that of the liquid, it, too, would just flow around the mount, and it would be nearly impossible to snag it.

    Gravitational pull and viscous drag

    Fd = 6π μ R v

    If the crystal’s density is larger than that of the liquid, the crystal will sediment under the influence of gravity toward the mount below it. The crystal’s sedimentation speed is determined by the balance between the gravitational force pulling down and the viscous drag force given by Stoke’s law that opposes motion.
    It’s an easy matter to show that this sedimentation speed varies as the square of the crystal diameter. Thus, the time you have to wait for a crystal to sediment onto the mount increases rapidly as the crystal gets smaller. A 10 μm crystal sediments at less than 10 μm/s, 100 times more slowly than a 100 μm crystal.
    In the videos below, the white teflon balls falling through glycerol differ in diameter by only a factor of two. How do their terminal speeds compare?

    The video to the left, in which we try to pick up a small white teflon ball in glycerol using a plastic spoon, illustrates the problem.

    Small teflon ball

    Medium teflon ball

     

    Dehydration

    Dehydration is a serious risk when working with very small crystals. Crystals smaller than 50 μm can dry out in seconds, degrading crystal diffraction. If you see diffraction spots that look like they come from salt, you know you’ve got a problem.


     

    Solutions

    Choose the right tool for the job

    With enough time and a steady hand, any mount style will do when dealing with small crystals. But to simplify and expidite your research process, Gentaur provides the best solution for handling small crystals with the Small Crystal Harvesting Kit. Reduce the problems associated with small crystals. Spend less time chasing them and eliminate dehydration issues. Included in this kit are:

    MicroMounts™ with a special wicking aperture that reduces laminar flow problems
    Low Viscosity CryoOil™ to store crystals and prevent dehydration prior to mounting
    MicroMesh™ to easily mount very tiny (<30 μm) crystals
    Reusable goniometer bases designed to decrease costs and increase throughput
    • Goniometer base holders for easy storage and cleaning
    Heavy–tweezers; ideal for handling mounts with reusable bases

    Small Crystal Harvesting Kit 

    Move slowly and carefully

    The presence of the mount’s aperture makes things a bit easier. As you move the mount through the liquid, liquid flows through the aperture. The crystal can flow along with this liquid and be sieved out. However, as the aperture gets smaller, the flow speed through it gets much smaller. If you move the mount too quickly, the liquid won’t have time to flow through the aperture. Your crystal may just flow around the aperture and end up on the neck of the mount.

    The key to mounting very tiny crystals, then, is to move the mount very slowly toward the crystal, allowing enough time for the crystal to sediment through the liquid toward the mount, and for liquid to flow through the aperture.
    This is illustrated in the video to the left, in which we retrieve a teflon ball in glycerol using fork.

    Dehydration

    To minimize dehydration, we recommend the following:
    1. Using Gentaur’s MicroMounts™, MicroLoops™, or MicroMeshes™ to quickly and easily mount your samples, minimizing the time for dehydration to set in
    2. Transferring your crystal to a drop of Low Viscosity CryoOil. Oils block evaporation during and after mounting
    3. Working in a humidified environment or in a cold room.
    o Evaporation rates are proportional to the quantity Δr.h. = 100% – r.h. so that increasing humidity from a typical laboratory value of 50% to 90% can reduce the evaporation rate by a factor of 5.
    o Evaporation rates plummet with decreasing temperature. The saturated vapor pressure of water at 4°C is 1/4 that at 25°c.



    Good luck!

    We hope this Tech Tip has given you further understanding about the complicated processes of crystallography. Please don’t hesitate to provide us with comments or suggestions at This email address is being protected from spambots. You need JavaScript enabled to view it.