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The new EazyReader® 7000
World's first 100 % scalable Elispot/Plaque Reader that may be upgraded on-site to a Single Cell/PBMC counter.
The core capabilities are:
- Single/Dual Elispot, visual Plaque and FOCI assay analyzer.
- All types of 96/384 well plates.
- Unique dual telecentric illumination with high intense power LEDs.
- Special morphological algorithms allow to separate clusters of spots/plaques/cells.
- 12 times Filter changer1 .
- Objective with Fixed Focal Length.
- 5 MPixel camera.
The upgrade options are:
- Zoom automation.
- Automatic objective changer with up to 4 objectives with 1 – 50 times magnification. Full field of view from 6 well plates to 400 x 400 μm.
- Up to 12 replaceable filters cubes for epi-fluorescence applications.
- Full power Fluoro LEDs with excitation wavelength between 350 - 730 nm.
- Multiple-segment scan of a well in up to 12x12 segments.
- Brightfield and Darkfield illumination.
- 12 or 25 MPixel camera, for higher magnification and larger camera field-of-view range.
1 Filter changer is empty for future applications.
Independence
The Bioreader® and EazyReader® operates with all commonly used Elispot/Fluorospot/Plaque/Fluoro-Plaque/CFU assays.
You can choose among all major assay manufactures.
Tests of antiviral substances
This Bioreader® supports testing and development of antiviral substances.
Example#1: The automatic analyses of Plaque reductions assay.
This assay applies diluted doses of a -possibly- antiviral drug into multiwell plates. The Bioreader automatically observes its capability to suppress the formation of plaques (-holes-) in the bacterial lawn.
The ‘Plaque reductions neutralization test’ (PRNT) is a variation of this test. It is an attempt to find an antibody that neutralizes a specific virus. This is a very valuable tool in antiviral vaccine research.
Examplel#2: The automatic evaluation of the Yield reduction assay
In several steps of operation cells are infected in presence of variable doses of a possibly antiviral drug substance.
Thereafter the virus titer is determined in the liquid by use of the plaque or TCID50 assay with of the Bioreader®.
The titer is calculated as the quotient of the positive cells/all cells, taking in account the virus dilution factor and the volume of the inoculum.
- Full automation of all mechanical functions incl. focus and zoom.
- Dual telecentric illumination for better cytokine quantification.
- Objetive with fixed focal length for 96 and 384 well plates.
- Or automated zoom for all other magnifications.
- Microscopic zoom system for microscopic cell counting.
- Hardware is automatically controlled by software measure setup (GxP-locked).
- No mercury or deuterium lamps required, thus 50T hourly lifetime.
- Improved suppression of auto-fluorescence, cluster separation, well recognition and cytokine volume quantification.
- New motor controller Gen. 7 with significant faster and more precise positioning.
- Single click export to PDF, Microsoft Office® applications.
- Front loader, automatic door.
- Interactive training program.
- Excel/Word/Power Point and LIMS export capabilities.
- Suitable for research and routine purposes.
- High contrast/low noise cameras.
- Optional full automation with plate 'feeding system'.
- DQ/IQ/OQ/PQ documention possible.
- 12 months warranty.
- EC declaration of conformity and EMC certificate available.
- Innovative Bioreader® and EazyReader® software combines ‘easy of use’ and versatility and flexibility.
- Scan, analyze and overlay live time.
- Creates up to 7 images for each well simultaneously during the scan.
- ‘Profiling’ app helps to create user independent measure protocols for Elispot beginners and references for experts.
- Verified in collaborative studies.
- Export options and reports with all scalable images and results even in one file.
- Customer specific export and report templates.
- Video clips and content specific help files.
- Qualified installation and training with each instrument.
- On-site or internet remote services and support.
- ‘Classified’ measure protocols ‘history’ tracking and comparison tools.
- User specific plates, Studies/projects, plate layouts/designs and measure protocols prevent from mix-up.
- More accurate ‘cytokine quantification’ based on the patented ‘photometric’ dual illumination system.
- Optional software features:
- 21 CFR part 11 based software module.
- Routine software incl. LIS/LIMS import and export.
Weight
Dimension
Fluorospot 1-4 colors
Multi Fluoro Color Reader,
best for Elispot and FluoroSpot
Applies ‘local neighborhood processing’ in order to compensate for uneven background on fluoro plates. This way the spot volume is quantified, independent from the local background.
Why use Fluorospot?
Enzymatic Elispot labeling
• allows to label 1-2 cytokines per cell simultanously.
• If you are interested to see ‘double secreting’ cells the stain of two cells in neighborhood may overlap. So, It is not clear if two cells are overlapping or one cells is really secreting two different cytokines (see below left).
‘Fluorospot’ labeling
Allows to label -a theoretical unlimited number different- cytokines. Because excitation for each cytokine is applied -one after the other-, decisions for double secreting cells is clearly.
One Image is acquired for each cytokine!
Currently Fluorospot assays with 1-4 Fluorophores are on the market.
Fluorospot offer's significant advantages over colorimetric formats, particularly in the areas of multiplexing and automated spot detection.
Moreover, as spot development is not enzymatic, signal intensity is directly proportional to the amount of analyte within.
SARS-COV-2
Vero cells infected by SARS-CoV-2
Inverted Immune fluorescence.
Secondary Antibody Cy3 AffiniPure Goat Anti-Human IgG
Bioreader® 7000 -F-Z-i micro
The versatile multiwell plate evaluation system for testing antiviral substances against SARS-COV2, HDV, hepatitis and other virus groups.
Many laboratories are currently researching the mechanisms by which viruses enter cells (with a focus on SARS-CoV-2).
Various proteins on the surface of the cells play a major role here. These are a present in different amounts from test person to test person.
The currently occurring coronavirus SARS-CoV-2 has the capability to penetrate the cell by the help of even small amounts of this specific proteins. Extensive research is required to develop substances that reduce or prevent the virus from entering the cell.
This is where the Bioreader® 7000 F-z-i Micro comes into play, which allows the researcher to test a large number of antiviral substances in vitro. The Bioreader® is currently used in leading national and international virological research laboratories with a large number of different cell lines and different viruses.
Another established method is the end-point limiting dilution assay for determining the mean Tissue Culture Infectious Dose (TCID50), which allows a statement to be made about how much the infection of the virus is slowed down or prevented by a substance.
In these and other in vitro tests, it is necessary to use extremely variable optical magnification so that plaques, cell clusters, foci as well as single cells are well imaged.
The cells / plaques / foci etc. must be countable from Petri dishes, cell culture bottles, 6-384 well plates and ultimately also microscope slides, both in white light and with various fluoro stimuli. With the Bioreader® 7000 -F-Z-i Micro the entire well /disk can be counted in full field of view or true optically enlarged partial areas.
The Bioreader® 7000 -F-z-i is ideally suited for these tasks and is already used in many laboratories for research into antiviral substances.
The Bioreader® 7000 -F-z-i Micro is characterized by the fact that it has three separate optical systems which are used automatically depending on the format used (Petri dishes / cell culture bottles, 6-384 multi-well plates, microscope slides). All measurement parameters (with over 100 setting parameters) are applied automatically, especially for the respective application.
Here, e.g. a differentiation between infected and non-infected single cells can be made.
Elispot in 384 microfilter plates
Small volume, higher throughput.
Elispot - enzymatic single color
Blue, red, green or silver substrat Elispot.
Detection antibody, enzyme-conjugate and precipitating substrate.
"The enzyme-linked immune absorbent spot (ELISpot) is a type of assay that focuses on quantitatively measuring the frequency of cytokine secretion for a single cell."
[https://en.wikipedia.org/wiki/ELISpot]
Why Elispot?
The cytokine Elispot assay is designed to enumerate cytokine secreting cells in single cell suspensions of lymphoid tissue, CNS tissue, bone marrow or preparations of peripheral blood mononuclear cells (PBMC).
The assay has the advantage of detecting only activated/memory T cells and the cytokine release can be detected at the single cell level, allowing direct determination of T cell frequencies.
The ELISPOT assay is an effective tool to enumerate antigen-specific T cells in the circulation of immunized humans and animals at much lower frequencies than possible with other currently available methods.
The ELISPOT assay has proven to be a sensitive and unique system to follow disease progression in human individuals or animals. Several studies have indicated that alterations in the frequency of cytokine pc in different compartments of the body adequately reflect changes in immune function.
The ELISPOT assay may be used to determine effects of drugs, chemicals or other compounds on cytokine secretion in vitro, thereby providing data on their putative modulatory effects on immune function in vivo
The ELISPOT assay is currently being used increasingly for the quantitative assessment of peptide reactive T lymphocytes from PBMC in infectious disease (3, 9) or in the course of vaccination trials aimed at the induction of tumor-specific T cells in patients with cancer.
Elispot may be used for Diagnosis of genetic defects,Allergic diseases,Autoimmune diseases,Transplantation,Cancer research,Acute inflammation,Acute infectious diseases and septic shock.
Elispot - enzymatic dual color
Blue and red Elispot substrate (mixed color violet)
Dual cytokine secretion.
Single cell infection assay
High Resolution Virus-infections-assay Analyzer.
Development of antiviral vaccines and substances.
For instance for CORVID, Hepatitis, Herpes, Adeno, ZIKA, Marek or Cytomegalovirus virus and more.
Immunohistochemical and Fluorophore or protein staining.
Liver, spleen or lung cells, Vero (kidney epithelial) , HEK(Human Embryonic Kidney) and more.
Single cell > 3 µ,Foci 10-2000 µ or Plaques 100-5000 µ,cell clones 50 - 2000 µ and more
Infection assay
GFP and Alexa 488 fluoro remission
Variable microscopic magnification.
Green fluorescence.
Adherent cells (primary human fibroblasts) in 96-well micro plates. Fluorophores: DAPI and AlexaFluor488.
RFP-expressed virus infected
"Using DNA recombinant technology, scientists combine the GFP gene to a another gene that produces a protein that they want to study, and then they insert the complex into a cell."
[https://embryo.asu.edu/pages/green-fluorescent-protein but with RFP instead of GFP]
GFP/DAPI-expressed virus infected
"Using DNA recombinant technology, scientists combine the GFP gene to a another gene that produces a protein that they want to study, and then they insert the complex into a cell."
Alveolar epithelial cells (AEC)
"Typically, type 1 alveolar cells comprise the major gas exchange surface of the alveolus and are integral to the maintenance of the permeability barrier function of the alveolar membrane. Type 2 pneumocytesare the progenitors of type 1 cells and are responsible for surfactant production and homeostasis."
[https://www.sciencedirect.com/topics/medicine-and-dentistry/alveolar-type-i-cells]
The alveolar epithelium is a major target in toxic exposures of the lung because of its structural delicacy and proximity to inhaled toxicants. Type II epithelial cells are important in maintaining the integrity of alveolar epithelium and normal lung function.
Unstained cells
AEC graylevels
"Typically, type 1 alveolar cells comprise the major gas exchange surface of the alveolus and are integral to the maintenance of the permeability barrier function of the alveolar membrane. Type 2 pneumocytesare the progenitors of type 1 cells and are responsible for surfactant production and homeostasis."
[https://www.sciencedirect.com/topics/medicine-and-dentistry/alveolar-type-i-cells]
The alveolar epithelium is a major target in toxic exposures of the lung because of its structural delicacy and proximity to inhaled toxicants. Type II epithelial cells are important in maintaining the integrity of alveolar epithelium and normal lung function.
AEC color
"Typically, type 1 alveolar cells comprise the major gas exchange surface of the alveolus and are integral to the maintenance of the permeability barrier function of the alveolar membrane. Type 2 pneumocytesare the progenitors of type 1 cells and are responsible for surfactant production and homeostasis."
[https://www.sciencedirect.com/topics/medicine-and-dentistry/alveolar-type-i-cells]
The alveolar epithelium is a major target in toxic exposures of the lung because of its structural delicacy and proximity to inhaled toxicants. Type II epithelial cells are important in maintaining the integrity of alveolar epithelium and normal lung function.
Live/dead
Live cells are white.
Dead cells are black.
Tryphaneblue stain.
Cell viability
6-CF appears as green fluorescence.
Live single cells
Bright field application Jurkatcells.
Live/apoptotic cells
NUCblue/interference/NUCgreen.
Mix of Jurkatcells untreated cell proliferate and TNF/CHX-treated that causes to apoptosis.
Transgene assay
"In its most precise usage, the term transgene describes a segment of DNA containing a gene sequence that has been isolated from one organism and is introduced into a different organism."
[https://en.wikipedia.org/wiki/Transgene]
Cells transfected with an expression plasmid encoding eGFP.
Cell compartment visualization and cell transfection efficiency
The Bioreader® 7000-F-z with micro-resolution can determine the transfection rate. If compartments are transfected with specific proteins and labelled with individual fluorophores they can be differciated.
MCMV (Murine Cytomegalovirus) greenfluoro remission
"Cytomegalovirus (CMV) (from the Greek cyto-, "cell," and megalo-, "large") is a genus of viruses in the order Herpesvirales."
MCMV (Mouse Cytomegalovirus) unstained
MCMV (Murine Cytomegalovirus) red fluoro remission.
"Cytomegalovirus (CMV) (from the Greek cyto-, "cell," and megalo-, "large") is a genus of viruses in the order Herpesvirales."
MCMV mutants got generated that express with tdTomato, eGFP or mCherry
MCMV mutants, tdTomato, Cherry, eGFP fibroplasts
HCMV (Human cytomegalovirus): unstained
"The glycoprotein gO (UL74) of human cytomegalovirus (HCMV) forms a complex with gH/gL. Virus mutants with a deletion of gO show a defect in secondary envelopment with the consequence that virus spread is restricted to a cell-associated pathway."
GFP immunofluorescence on HEK 293 cells
"Human embryonic kidney 293 cells, also often referred to as HEK 293, HEK-293, 293 cells, are a specific cell line originally derived from human embryonic kidney cells grown in tissue culture."
NK92 cells natural killer cells (human cell line) same samples microwell plate.
"Natural killer cells, or NK cells, are a type of cytotoxic lymphocyte critical to the innate immune system. Typically, immune cells detect major histocompatibility complex (MHC) presented on infected cell surfaces, triggering cytokine release, causing lysis or apoptosis."
NK92 cells natural killer cells (human cell line) expressing green fluorescent protein (gfp)
"Natural killer cells, or NK cells, are a type of cytotoxic lymphocyte critical to the innate immune system. Typically, immune cells detect major histocompatibility complex (MHC) presented on infected cell surfaces, triggering cytokine release, causing lysis or apoptosis."
Mouse spleen cells mostly lymphocytes stained with FITC-conjugated anti mouse cd45 stain
"The spleen is an organ found in virtually all vertebrates. Similar in structure to a large lymph node, it acts primarily as a blood filter. A study published in 2009 using mice found that the red pulp of the spleen forms a reservoir that contains over half of the body's monocytes." [https://en.wikipedia.org/wiki/Spleen]
Foci assay: Very large Foci
"The focus forming assay (FFA) is a variation of the plaque assay, but instead of relying on cell lysis in order to detect plaque formation, the FFA employs immunostaining techniques using fluorescently labeled antibodies specific for a viral antigen to detect infected host cells and infectious virus particles before an actual plaque is formed."
Foci assay: total well view
"The focus forming assay (FFA) is a variation of the plaque assay, but instead of relying on cell lysis in order to detect plaque formation, the FFA employs immunostaining techniques using fluorescently labeled antibodies specific for a viral antigen to detect infected host cells and infectious virus particles before an actual plaque is formed."
CV Plaque assay.Example with large and fuzzy plaques
Cells were infected and covered with an overlay. Cells stained with CV survive. Unstained cells were killed by the virus.
TCID50 viral assay
Cells were infected and covered with an overlay. Cells stained with CV survive. Unstained cells were killed by the virus.
Plaque assay extremely large spots
"The TCID50 (Median Tissue Culture Infectious Dose) is one of the methods used when verifying viral titer. TCID50 signifies the concentration at which 50% of the cells are infected when a test tube or well plate upon which cells have been cultured is inoculated with a diluted solution of viral fluid."
Neutralization assay
"The plaque reduction neutralization test is used to quantify the titer of neutralizing antibodies for a virus. The serum sample or solution of antibodies to be tested is diluted and mixed with a viral suspension."
[https://en.wikipedia.org/wiki/Plaque_reduction_neutralization_test]
Plaque assay: very bright plaques in 24 well plate
Cells were infected and covered with an overlay. Cells stained with CV survive. Unstained cells were killed by the virus.
Detection of Marek's disease virus Plaques after staining with specific anti-MDV antibodies and Alexa 488 labelled secondary antibodies
"Marek's disease is a highly contagious viral neoplastic disease in chickens. The disease is characterized by the presence of T cell lymphoma as well as infiltration of nerves and organs by lymphocytes. Viruses related to MDV appear to be benign and can be used as vaccine strains to prevent Marek's disease."
Detection of Marek's disease virus Plaques after staining with specific anti-MDV antibodies and Alexa 568 labelled secondary antibodies
"Marek's disease is a highly contagious viral neoplastic disease in chickens. The disease is characterized by the presence of T cell lymphoma as well as infiltration of nerves and organs by lymphocytes. Viruses related to MDV appear to be benign and can be used as vaccine strains to prevent Marek's disease."
Organoids
"An organoid is a miniaturized and simplified version of an organ produced in vitro in three dimensions that shows realistic micro-anatomy. The technique for growing organoids has rapidly improved since the early 2010s."
Dual Transgene assay
Transgen:
A segment of DNA introduced to some other organism.
Adenovirus
"Adenoviruses (members of the family Adenoviridae) are medium-sized (90–100 nm), nonenveloped (without an outer lipid bilayer) viruses with an icosahedral nucleocapsid containing a double stranded DNA genome.
Their name derives from their initial isolation from human adenoids in 1953.
They have a broad range of vertebrate hosts; in humans, more than 50 distinct adenoviral serotypes have been found to cause a wide range of illnesses, from mild respiratory infections in young children (known as the common cold) to life-threatening multi-organ disease in people with a weakened immune system."
Various media/strain combinations on petridishes
