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Immunology

The development of new vaccines and therapeutic antibodies forms only a small part of what can be carried out in the field of Cellular Immunology with SYNENTEC's automated cell imagers.

Introduction

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SYNENTEC supports your work in various fields of immunological research. Our automated cell imagers are suitable for many areas of Immunology, e.g.:

  • Humoral Immune Response (Antigen-antibody-reaction, IgG antibodies, Lymphycytes, Adapted Immune Answer…)
  • Infectiology (e.g. studying Trojan Horse Mechanism)
  • Immune Deficiencies (Allergies, Autoimmune Diseases such as Diabetes Mellitus Type I, Hashimoto Thyreoides, Rheumatoid Arthritis, Celiac Disease, AIDS…)
  • Vaccine Development
  • Immune Cyto Chemistry (e.g. CD-marker Detection ...)
  • Therapeutic antibodies (for example Cancer Therapy…)
  • Reproductive Immunology

Applications such as fluorescent antibody-staining, apoptosis monitoring, toxicity-studies, surface marker analysis and growth curve generation represent only a small part of the applications in which SYNENTEC actively supports your daily lab work with their fully automated high-throughput cell imagers Cellavista® and NyONE®. For further information about assays like toxicity tests, apoptosis monitoring or viability tests visit the Drugs & Cancer or the Cell Line Development area of this homepage.

Immuno Cyto Chemistry (ICC/IHC) – Multicolor Cell Analysis

[Virtual Cytoplasm 2F]

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By immunocytochemistry, proteins or other structures can be made visible with labeled antibodies. If the antibodies are labeled with common fluorescent dyes, these markers can be analyzed with SYNETEC's cell imagers. Furthermore a detection of immunoprecipitation (e.g. with HRP and DAB reagent) can also easily be performed.

Using ICC/IHC, it can be determined for example, in which tissue a target protein is present (e.g. tumor analysis) or in which compartment of the cell it is localized.

For antibody staining fixed tissue and cells can be used, but also live-cell staining is measurable with our cell imagers NyONE® und Cellavista®.

Our fluorescence detection is very sensitive so that even weak stained antigens can be detected and the YT-image processing software® can even analyze the labeled cells unwashed samples with a high fluorescent background signal.

Extract from the software readout (for further information look at ShortNote Virtual Cytoplasm 2F)

  • total # of Cells
  • % of Nuclei stained Cells
  • % of Nuclei and AB-stained Cells

CD Marker – Immunological Staining Of Cluster Of Differentiation

[Antibody binding]

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Cluster of differentiation (CD) are a large family of cell surface markers. The differentiation is conducted by functional and biochemical criteria. Their localization on the cell surface explains their main functions as cell adhesion proteins, signal transducer, receptors, activators, apoptosis initiators etc. and enables an easy immunological staining on living cells.

Since the CD markers are very cell type specific, there is a lot of ongoing research involved in the development of antibodies (AB) against CD-markers expressed by pathological cells. A good example is Retuximap, a monoclonal AB against B-cell marker CD20, which applies in B-cell derived lymphomas and leukemia but also in B-cell mediated autoimmune disorders.

To check the fluorescence filter specifications of our systems visit the Fluorescence Viewer.

Extract from the software readout for suspension cells (for further information look at ShortNote Antibody binding)

  • # of objects
  • Mean size of objects
  • Mean intensity of objects
  • Total fluorescence per well

Viral plaque Assay – Influence Of Viruses

[Plaque Assay]

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The viral plaque assay is a method for the detection and quantitation of infectious cytopathic viruses which are added in different dilutions in a cell layer.

The difficulty is to find lysed cell areas between a confluent cell monolayer - the plaque center may miss out cells due to virus-induced lysis.

The high-resolution, precision optics of SYNENTEC's cell imagers is very well suited for this type of virus analysis. For standardized and quick evaluation of this assay SYNENTEC developed the viral plaque operator.

Extract from the software readout (for further information look at ShortNote Plaque Assay)

  • # of Plaques
  • Plaque Area [%]
  • Average Plaque size [µm2]

Focus Forming Assay – Virus Titer Quantitation

[Dark spots]

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The focus forming assay (FFA) is an immunostaining technique and a variation of the viral plaque assay. Instead of detecting the plaque formation after virus-induced cell lysis these assays detect infected host cells and infectious virus particles before a plaque is formed.

Different cell monolayer samples were infected with a serial dilution of the virus of interest. After incubating the samples overnight the host cells built clusters of infected cells which could be visualized with specific horse radish peroxidase (HRP) coupled antibodies against a viral antigen.

The HRP can oxidize with the 3-3’diaminobenzidine tetrahydrochloride (DAB) which results in  brown/black insoluble precipitates. These foci were visualized with the brightfield illumination and the number of foci was quantified with our YT-image analysis software®.

Extract from the software readout (for further information look at ShortNote Dark Spots)

  • # of Foci
  • mean Foci Size
  • total Confluence
  • % Foci covered Area

Cell Proliferation – Keep An Eye On It

[Confluence – Suspension Cell Count]

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Confluence monitoring is a useful tool to determine various properties of your cell line. SYNENTEC’s confluence image processing analysis is capable to solve a vast range of different questions – e.g. to monitor the growth rate under selective conditions or to prove toxicity by adding an antibody and monitor the growth rate over different concentrations as a first step of proving the influence on the cell type of interest.

With SYNENTEC’s automated cell culture microscopes NyONE® and Cellavista® and the included YT-image analysis software® the proliferation analysis is possible for a variety of cells. Adherent lines can be detected as well as suspension cells. In addition, it is very easy to quantify additional fluorescent labels and their ratio to the total growth area.

Extract from the software readout for adherent cells (for further information look at ShortNote Confluence / Confluence 1F)

  • % Confluence BF
  • % Confluence FL
  • % Ratio of Confluence BF/FL

Extract from the software readout for suspension cells (for further information look at ShortNote Suspension Cell Count / Suspension Cell Count 1F)

  • # of Cells in Brightfield
  • # of Cells in Fluorescence
  • # of Cells Brightfield AND Fluorescence

IgG-Quantitation – Sometimes Less Means More

[PAIA]

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During the cell line development process hundreds to thousands of clones have to be screened to find the most promising, i.e. well growing clones that are also good producers for further development. The process of single cell cloning is usually performed in 96-well formats and increasingly also in 384-well plates. Thus, there is limited sample volume available for analysis and it is desirable to get the results as quickly as possible.

PAIA BIOTECH has taken on this challenge and their scientific team has developed a new ultra-fast-low-volume assay for IgG-quantification directly from the supernatant of the culture vessel!

Assay Principle

Protein A coated capture beads, analyte and fluorescence markers are incubated in the wells of a 384-well PAIAplate. After sedimentation of the beads with the bound analyte-marker-complexes, the remaining unbound fluorescence marker is measured with the NyONE® or Cellavista®.

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Features

  • High throughput in 384 well plates
    • processing time for full plate  < 1 hour
  • Low sample consumption
    • 2- 10 µL of crude sample
  •  Simple protocols in single-use plates
    • no washing , no blocking, no regeneration
  • Cost-efficient (No Capex)
    • runs on fluorescence microscopes

Software readout

PAIA Biotech has developed a software tool for the rapid generation of calibration curves, calculation of sample concentrations and result exports. The data generated with our YT-analysis software® can be exported as csv-files and can be imported into PAIAs software tool.

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