Xenometrix
Ames Mutagenicity Assays
Cytotoxicity Assays
Why should I perform the Ames II / Ames MPF Mutagenicity Assays rather than the standard plate incorporation test?
With the ready-to-use reagents and media, media preparation, autoclaving and sterility testing is no longer necessary. The Ames II / Ames MPF Mutagenicity Assays are liquid microplate modifications of the standard Ames test which offer a higher speed format, easier handling and the possibility of automated plating and plate reading. The assays are fast and efficient, show good correlation with the standard Ames test and consume less test compound and consumables than the standard Ames test.
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Do you offer presentations of your tests in our facilities?
We are happy to discuss your requirements and make suitable arrangements for you. Please contact us directly to discuss this.
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How long does it take to perform the Ames II / Ames MPF Mutagenicity Assays?
The assays last two and a half days, however with much less hands-on-time as compared with the standard Ames test.
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How much test compound is needed?
With a top dose of 5 mg/ml, 6 concentrations, triplicate, a ½ log dilution range, 2 strains ± S9, the lowest amount of test compound is 26.5 mg. With a top dose of 1 mg/ml, 6 concentrations, triplicate, a ½ log dilution range, 2 strains ± S9, the lowest amount of test compound is 5.3 mg.
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Which bacterial strains are available for the Ames II / Ames MPF Mutagenicity Assays?
In the Ames II Salmonella typhimurium TA98 is used for the detection of frameshift mutations and TAMix for the detection of base pair mutations. TAMix is a mixture of the strains TA7001, TA7002, TA7003, TA7004, TA7005, TA7006 in equal proportions. Individually these strains are designed to revert by only one specific base-pair substitution. With this mixture, all the possible base pair substitutions can be represented in one culture.
In the Ames MPF series of mutagenicity tests the following S. typhimurium strains recommended by the OECD guideline 471 are available: TA98, TA100, TA1535, and TA1537. Two E.coli strains also mentioned in the guideline 471 are also available in the Ames MPF format: E. coli wp2 uvrA and E. coli wp2 [pKM101].
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Are there publications with the Ames II / Ames MPF assays?
Please refer to our Literature section.
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Can we send you compounds for testing?
Yes, we do test compounds for customers with any of our strains or their combination. A Secrecy Agreement may be signed for commercially sensitive information and materials.
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Are the strains, reagents and media available separately?
Yes, all the necessary components for performing the Ames II /Ames MPF Mutagenicity Assay are also available separately.
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How many compounds can I test with your kits?
The Ames II / Ames MPF Mutagenicity Assays are available in 2 sizes with enough bacteria and media for either 1 or 10 samples. All kits allow to test the indicated number of samples in triplicate at 6 concentrations, with negative and positive controls.
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Can you provide S9 / positive control chemicals / plastic ware?
We also provide S9 fraction and positive controls, and if needed the necessary plastic ware for the 1-sample kits. (For reasons of cost effectiveness we do not sell larger amounts of plastic ware.)
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Sometimes when I am scoring my 384-well plates, there are wells that look like they are starting to turn from purple to yellow, but are not completely yellow. Should these be scored as positive or negative?
Any color change away from the purple of negative wells should be considered positive. Often (but not always) a small colony of bacteria is visible in positive wells. We recommend to use a light box in order to better see the changes in color and the colonies.
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I have problems with bubbles in the wells. They give me unreliable OD readings.
It is essential to remove all bubbles before reading the OD of the wells. This can be achieved by passing a gas flame over the wells. You can use an inverted Bunsen burner or a mini torch. Be careful not to overheat the wells or to burn the plastic. Be careful for the presence of flammable materials or liquids next to your workspace. You could also use a hair dryer for this purpose.
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I observe strong variations of OD readings between replicate wells.
Possible sources of variation are: cell clumps resulting in uneven cell numbers in the wells; partial loss of cells during washing steps; uneven pipetting of reagents (loose tips). Be particularly careful for the presence of air bubbles in your wells. Remove them with a gas flame (Caution!) or a hair dryer.
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What solvents up to what concentration can I use in the tests?
This depends also on the cell line and the length of incubation. If your substance is not soluble in water we recommend to try DMSO. It is safe to use in most systems up to 2%. If higher concentrations need to be used you should first test the solvent effect compared to a blank. Ethanol or methanol can be used as a solvent, tool. Again, 2% should be fine.
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Do I have to use such strong acids like 12N H2SO4 or 37% HCI at the end of the Glucose test?
Yes, an optimal color development requires the use of strong acids.
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In the Glucose test I see very little difference between the medium control and the positive control with cells. What can I do to increase this difference?
First, make sure you dilute your samples when using media with high glucose content. The assay can measure glucose concentrations in the range of 1 - 200 μg/ml.
You can increase the lenght of incubation of the cells and/or the number of cells per well. It can take several days until cells have consumed 50% of the available glucose. As a rough indication you can observe the color of the medium: when it turns yellow the cells have most likely also consumed a significant part of the total glucose.
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The reconstituted glucose working solution has turned slightly pink after storage at 4°C for several weeks. Can I still use it?
Yes, as long as no precipitate has formed you can still use the solution. The absolute OD readings will be slightly higher, but the OD differences should be about the same.
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Can I perform the kinetic OD reading also at RT?
Yes, the kinetic reading can also be done at RT. You should extend the reading to 1 hr.
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My plate reader does not allow to read at 340 nm. Can I use an alternative wavelength setting?
No, you need a plate reader capable of reading plates at 340 nm.
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Why is there a slow decrease of OD340 WIth time even in the medium control?
Fetal Calf Serum used in the medium is a (usually small) source of LDH activity. If this activity is too high, you may try to reduce the amount of FCS in your medium or switch to another FCS source.
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Can I store the supernatant and run the assay at a Later time?
Yes, you can store the plate at 4°C for a few hours, provided it is sterile and you have a protein source in your medium (FCS, BSA). We do not recommend freezing of the supernatant. Be sure the supernatant and the assay plate have room temperature before performing the assay.
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What kind of cytotoxic substances can be measured by the test?
The test is particularly suitable for substances affecting the intergrity of the cell membrane.
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Can I directly compare the results (units per ml) obtained with results obtained with a different LDH assay?
No, enzymatic activities measured as units per volume depend on experimental conditions (e.g. buffer composition, temperature, substrate concentrations). If you want to compare results with an other test you could run the same substance in both tests and see how the results correlate.
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Pyruvate is often present in culture media such as Dulbecco’s, Iscove’s or Ham -F12. In the test kits of competitors it can interfere with the assay - does it also interfere in the LDHe assay by Xenometrix?
In the Xenomtrix version of the assay the conversion of pyruvate to lactate by LDH is measured. A defined amount of pyruvate is added to the reaction mixture which is about 50x higher than the contribution by the medium. Therefore, pyruvate present in culture media does not significantly contribute to the reaction equilibrium or reaction rate.
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PAC IV Stop Solution has developed a dark precipitate. Can I still use it?
You can remove the precipitate by centrifugation at 3000 rpm for 5 minutes and sill use PAC IV.
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XTT and MTT both detect mitochondrial metabolism. Which test should I choose and why?
XTT has two main advantages over MTT: It is more sensitive, and the reaction results in a soluble formazan dye. This eliminates a final solubilization step which means less manipulation and consequently a reduced risk of error.
MTT has the advantage of being less expensive.
Another consideration may be the filter settings available with your plate reader: XTT requires 480 or 450 nm plus a reference filter at 690 nm; MTT requires 540 nm plus a reference filter at 690 nm.
If available filters are no constraint in your lab we recommend to use the XTT test.
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U-CyTech
The ELISPOT assay - general
The ELISPOT assay - specific
What is a cytokine ELISPOT assay?
The cytokine enzyme-linked immunospot (ELISPOT) assay is a versatile and highly sensitive method for the ex-vivo quantification of cytokine secreting T cells after stimulation with an antigen in vitro. In fact, each cell can be detected by the assay as long as a characteristic protein is released and specific antibodies are available.
The ELISPOT assay is very similar to an ELISA and is based on the same immunochemical 'sandwich' principle. The major difference is that an ELISPOT is a combination of both an immunoassay and bioassay because living cells are cultured directly in the wells of the ELISPOT plate. In the first step, anti-cytokine capture antibodies are immobilized onto the solid phase. Activated T cells are subsequently brought into the wells and incubated for a certain length of time. The released cytokines are trapped by the capture antibody in the immediate vicinity of the producing cells and then visualizedby a combination of a second antibody coupled to an enzyme and a chromogenic substrate. A single cell forms a colored 'footprint' (spot) on the bottom of the plate representing its cytokine secretory activity. The frequency of spot forming cells is then estimated from the number of spots in the well and the cell input. The term 'spot-forming cells' or SFC, is used as a quantitative measure for the number of cytokine secreting T cells in the ELISPOT assay.
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Why choose an ELISPOT assay?
ELISPOT assays are among the most-sensitive and -specific methods available for cytokine research permitting the ex-vivo identification of cells actively secreting cytokines. Moreover the assay is fast and highly efficient and can detect a single cell out of a million. Major advantages of the ELISPOT assay are its relatively easy performance, its potential for high throughput screening and no requirement for expensive instruments. Because of the short-term in vitro culture, the measured response closely mirrors the in vivo T cell frequency of an individual.
The ELISPOT assay has the lowest detection threshold among the assays available for detection of T cell responses. The classical T cell assays are lymphoproliferation assays (LPA) and cytotoxic T lymphocyte (CTL) assays that measure CD4+ and CD8+ T cell-mediated immune responses, respectively. Both the CTL and LPA assays have their drawbacks including the use of radioactivity, low throughput screening, decreased sensitivity in cryopreserved specimens and technical burden.
ELISPOT assays has great potential as a diagnostic tool. It is believed that the ELISPOT assay is currently the best suited assay for evaluation of CD8+ T cell responses on the scale of phase I/II trials involving up to a few hundred volunteers. Even in phase III trials, which will be focused on the efficacy of the vaccination rather than on immunogenicity and will enroll several thousands volunteers, either the fresh- or cryopreserved-cell ELISPOT assays may have their place for cross-sectional immunogenicity reference.
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How does an ELISPOT assay differ from an ELISA?
An ELISA determines the total concentration of the secreted cytokine, whereas an ELISPOT enumerates cytokine secreting cells answering the question 'what is the frequency of secreting cells?'. Therefore ELISPOT should be used not 'instead of ' but rather 'in addition to' ELISA. An ELISPOT assay can be up to 400 times more sensitive than a conventional ELISA because the cytokine is captured directly onto a plate before having the change to be diluted in the culture supernatant, degraded by proteases or captured by cytokine receptors on adjacent cells.
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In which area of research should an ELISPOT assay be used?
The ELISPOT assay can be used in many areas of research such as cancer, infectious disease, autoimmune disease, allergy and organ transplantation. The assay is particularly effective for the measurement of antigen-specific responses post-vaccination in peripheral blood cell preparations.
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What type of information can be obtained from an ELISPOT assay?
One key piece of information that can be obtained from ELISPOT assays is the frequency of antigen-specific T-cells within a pool of different cell types. This frequency reflects the clonal size of the antigen-specific T-cells and, therefore, the magnitude of T-cell immunity.
Example of human IFN-gamma ELISPOT results:
2x10E5 human PBMC/well
PBMC stimulated with 0.1 µg/ml L. major antigen
Upper: Leismania patient
Lower: Healthy individual
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Are there special requirements and storage conditions for blood?
Whole blood should be kept at room temperature (RT) until processing. Do not refrigerate. If collected elsewhere, samples should be shipped at ambient temperature to the desired laboratory. Samples should not be at RT for more than 24h after draw.
The recommended anti-coagulants are citrate and heparin. For both substances there are no reported adverse effects on cellular function in the ELISPOT assay. EDTA, on the other hand, inhibits coagulation through calcium chelation which may impair cytokine induction during antigen-specific re-stimulation and is therefore dissuaded for use as an anti-coagulant.
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What should I know about cells?
Monocytes are critical as antigen-presenting cells (APCs) and a low number of monocytes can lead to lower spot forming cells (SFCs). Particularly cryopreserved samples show a decreased number of APC. Replenishment of monocytes may therefore be necessary. The presence of dead cells can impact the functioning of the assay and a cut-off of > 80% viability is recommended as a minimum. In some cases, even though the number of dead cells is low, there may be no spots formed because of apoptosis.
Example of human IFN-gamma ELISPOT:
2x10E5 PBMC/well
Upper: monocyte depleted sample
Lower: normal sample
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How many cells should be put into each well?
ELISPOT sensitivity critically depends on high cell density. A high number of cells enhances the probability of contact between stimulating and responding cells. For antigen-specific responses, optimal stimulation of T cells can be achieved when the total cell number approaches 5x10E5 to 10E6 cells/well/ml. In a 96-well ELISPOT plate, a maximum of 2x10E5 cells can be put into each well because this number of cells forms a tight monolayer on the bottom of the wells. Higher numbers of cells will lead to piling up of cells and linearity between cell input and detected spot frequency is lost. In general, 2x10E5 PBMC generate less than 100 antigen-specific SFC per well. For polyclonal stimuli such as mitogens or PMA + ionomycin, 10-100-fold less cells should be plated (2x10E3 to 2x10E4) to obtain individual spots at a spot frequency of 50-100 SFC/well. Too many spots will lead to overlap of individual spots and high background staining.
Example of Mouse IFN-gamma ELISPOT:
Stimulus: concanavalin A
Upper: 2.5x10E4 mouse (Balb/c) splenocytes/well
Lower: 1x10E5 mouse (Balb/c) splenocytes/well
(resulting in too many spots per well)
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How long should I incubate the cells?
A 24-48h preincubation step at high cell density (>10E6 cells/well/ml) is required when full-length proteins or peptide pools of 15 mers or longer are used for re-stimulation. These antigens must first be internalized, processed and presented by antigen-presenting cells (APCs) via MHC class I/II molecules before they can stimulate cytokine release by T cells. Omitting this step leads to a significant lower frequency of spot forming cells (SFCs). On the other hand, small (synthetic) peptides (8-10 mer) can directly be presented by APC to CD8+ cells and consequently need no preincubation step.
For more information please download our Addendum which contains guidelines and troubleshooting for ELISPOT analyses.
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How critical is the culture medium?
For the 'direct' human ELISPOT assay (no preincubation step), serum-free medium (AIM-V) has proven to be the best choice for obtaining an optimal T cell response. On the other hand, the ‘indirect’ assay (with preincubation step) requires medium (like RPMI-1640) supplemented with 10% FCS or human serum. AIM-V medium should not be used in the ‘indirect’ assay.
It should be mentioned that some batches of fetal calf serum (FCS) or human serum may non-specifically activate the cells and can therefore be the cause of background spot formation. Different batches of FCS and human serum should therefore be pre-tested before using it in the ELISPOT assay.
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What about a positive control?
There are several possibilities, but antigen-specific stimuli are preferred over polyclonal stimuli such as mitogens and anti-CD3/CD28 antibodies. For the human system, vaccin proteins (Tetanus toxoid, Hepatitis B, etc) or a pool of synthetic peptides of common human virus epitopes are excellent positive controls often producing a high number of spot forming cells. When using vaccin proteins, it should of course be known whether blood donors have ever been vaccinated and how long ago.
For more information about stimuli please download our Addendum which contains guidelines and troubleshooting for ELISPOT analyses.
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Is there a method to decontaminate the plate when using infectious agents?
In case cells in the transparent polystrene-bottomed ELISPOT plate are contaminated with infectious agents such as enveloped viruses (e.g. HIV, SIV), the wells can be decontaminated after the preferred incubation time by first removing the cells from the well, adding 100 µl 1% Triton X-100 and then to led it stand for 30 minutes at room temperature. Thereafter wells can be safely washed as usual.
When PVDF membrane-bottomed ELISPOT plates are used, they can be decontaminated after the preferred cell incubation time by first removing the cells from the well, washing 5 times with PBS, adding 200 µl 4% Paraformaldehyde and then to led it stand for 5 minutes (max) at room temperature. Thereafter wells can be safely washed as usual. These procedures may lead to a slightly higher background staining but has no adverse effect on spot number and size.
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What about antibodies?
Selection of optimal primary and secondary antibodies in an ELISA system does not imply that these are also the best antibodies for an ELISPOT assay. The best antibody pairs are selected by the manufacturer on basis of extensive research and should be used as a matched antibody pair for the ELISPOT.
One of the most important parameters to standardize is the total amount of antibody used for coating. A general guideline is that approx 0.5-1 µg per well of coating antibody result in well-defined spots. Coating concentrations that are too low result in not only fainter spots but also fewer detectable spots. Therefore, the most efficient concentration needs to be established to concur with cost-effectiveness without compromising spot quality and ultimately spot count.
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How critical are the incubation times mentioned in the ELISPOT manual?
For optimal results, the coating antibodies should be left in the wells of the ELISPOT plate for minimal 16 h at 4°C. The blocking buffer, detector antibodies and conjugate can be incubated for 1 h at 37°C, 2 h at room temperature or overnight at 4°C without any problem.
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Once the plates have been coated, how can they be stored and for how long?
Antibody coated plates (both polystyrene- and PVDF membrane-bottomed) can be prepared 7 days ahead. Seal the plate against evaporation and store these plates at 4°C. Be aware that for long-term storage, sterility should be guaranteed. Moreover, it is recommended that after coating, the wells are washed with sterile PBS and blocked with Blocking solution. This blocking solution is left in the wells until use.
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What type of spot detection systems are available?
Horseradish peroxidase (HRP), alkaline phosphatase (AP) or gold particles can be used as streptavidin conjugates. The advantage of using HRP is its fast turnover rate (spots develop fast), whereas the drawback is increased background. The HRP substrate AEC (3’-amino-9-ethylcarbazole) forms intense red colored spots. However AEC is unstable and spots will bleach in a short period of time.
AP has a linear reaction rate with little risk of high background. However spot development is slow and bleaching of spots is a problem.
A highly effective way of spot staining can be achieved with gold-labeled streptavidin using silver precipitation for spot visualization. Because of the high stability of silver, spots do not fade and ELISPOT plates can be reanalyzed after being stored for several years at room temperature.
Example of Human IFN-gamma ELISPOT:
2x10E5 PBMC/well; stimulus: viral peptide pool
Upper: Silver staining (transparent polystyrene-bottomed well)
Lower: AEC staining (PVDF membrane-bottomed well)
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What are the characteristics of a spot?
Each spot within an ELISPOT well is the ‘footprint’ of a single cell that has released a relatively high amount of cytokines. True spots have a dense center with a light outer ring caused by the diffusion of the cytokine from the producing cell.
The color depth or the size of spots depends on the amount of secreted cytokines. Only strong and well-defined spots should be counted; any small or faint spot is likely to be an artifact and should be ignored.
Artifactual spots can be caused by the aggregation of antibodies used in the process of coating and detection. Also incomplete removal of cells from the plate after incubation can be a source for artifactual spots. A simple measure to minimize false spot formation is to thoroughly wash the plate between all incubation steps and after completion of the coloring reaction.
Sometimes small and very dark spots appear in the background that are not generated by secreted cytokines. In the visual evaluation, these spots are differentiated from ‘true’ spots by their sharper edges. True spots always have a dark center with fading color intensity towards the edges. These dark spots have a homogeneous intensity and may originate from small cell membrane fragments firmly attached to the solid support carrying receptor bound cytokines.
The average size of a true spot is 30-150 µm, although this varies depending on incubation time, microtiter plates, antibody source and concentration, enzyme activity, substrates and other materials used as well as the functional state of the cytokine-secreting cells.
Manual counting of spots by light microscopy is a laborious task and strongly dissuaded in case many wells need to be analyzed. The availability of sophisticated computer-controlled ELISPOT readers from different companies offers a complete solution for precise and automatic evaluation of ELISPOT data and offers various procedures to overcome variable background intensity problems and to distinguish true spots from artifacts by image analysis.
In all cytokine ELISPOT assays, a wide spectrum of spot sizes and densities can be seen. Thus when analyzing ELISPOT results, cut-off values need to be set for the minimum spot size to be counted. The maximum spot size must likewise be defined so that clusters of cells can be identified as such. The minimum and maximum ‘gates’ set will critically affect the number of spots counted. For this reason, one of the main goals of ELISPOT image analysis has been to establish absolute criteria for gating.
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Is there an explanation for the stripy appearance of some spots?
Certain stimuli trigger the release of chemokines which causes the T cells to move around during incubation producing a cytokine trail in the well. This is particularly true when T cells are activated with polyclonal stimuli such as mitogens and anti-CD3/CD28 antibodies. Also the movement of plates during cell incubation may cause the cells to roll generating irregular or oblong-shaped spots.
Picture:
Chemokine mediated spurring of spots
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What are the small, dark spots that are not generated by secreted cytokines?
There are two possibilities. Due tot static electricity, dust particles attach to the underside and/or inside of the well of the transparent polystyrene-bottomed plates. Upon visual evaluation, these spots vary in size and are highly irregular. By cleaning the underside of the well with 70% ethanol, followed by blowing 4-5 bar compressed air against the underside and into the well, this problem can easily be solved.
The second possibility are cell wall fragments containing receptor-bound cytokines that stick to the coating antibodies in the well. These particles are very small, have sharp edges and cannot easily be removed by standard washing procedures. Remarkably, only PBMCs from certain individuals generate these ‘sticky’ spots. By proper gating, these small artifactual spots are ignored by standard ELISPOT readers.
Pictures:
Upper: example of dust particles
Lower: example of spots generated by cell wall fragments
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What type of plate can I use?
ELISPOT assays can be performed using either 96-well clear polystyrene-bottomed wells or membrane-bottomed wells such as PVDF and nitrocellulose. Both solid supports binds proteins by hydrophobic interactions (Van der Waal’s forces).
Membranes have a higher retaining capacity for antibodies because of a higher surface area than polystyrene plates. However, this increased surface area is not required for ELISPOT analyses since there is no flow of reagents through or across the membrane but rather a diffusion of cell-secreted molecules towards capture antibodies immobilized on top of the membrane. In fact, all of the antibodies immobilized on the top surface probably has a maximum penetration of 1-2 microns in depth. This explains that for both polystyrene- and membrane-bottomed plates the optimal concentration of the capture antibody for coating is similar. It should be stressed, however, that only a very few types of 96-well polystyrene plates have a sufficient high antibody binding capacity for use in ELISPOT analyses.
There are both sterile and non-sterile microtiter plates available for ELISPOT analyses. Since ELISPOT requires short culture times and includes the use of antibiotics in the culture medium, non-sterile plates can be used without complication.
A disadvantage of using PVDF membrane-bottomed plates is that for optimal binding of the capture antibody a prewetting step with 70% ethanol is required. Within 1-2 minutes after prewetting, the membrane should be rinsed thoroughly by adding coating buffer to the well and aspirating or decanting immediately thereafter. The rinse step should be repeated three times to efficiently wash out the ethanol before the solution with capture antibody is added to the wells. This prewetting step creates the risk of liquid collecting under the membrane. This liquid cannot be washed out effectively and may create serious problems later in the ELISPOT assay such as high and irregular background staining. Traces of ethanol trapped under the membrane may even suppress or completely inhibit cytokine release. Polystyrene-bottomed plates do not have such problems.
Because the polystyrene-bottomed plates are far more cost-effective than PVDF membrane-bottomed plates and the fact that polystyrene-bottomed plates in combination with silver staining show a similar or even higher sensitivity, it might be desirable to switch from PVDF membrane- to polystyrene-bottomed plates. However switching plates requires a revalidation of the protocol and possibly involves protocol changes. The same cells should be run in the same experiment on both plate types and several of such experiments should be performed.
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What kind of pipette tips should I use?
For the pipetting of cells it is important to use tips with a wide opening to avoid shearing stress upon the cells that may induce apoptosis and thus inhibition of spot formation.
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What about washing?
A critical issue in ELISPOT analyses, is the wash procedure. Washing of ELISPOT plates is necessary to remove cells and residual biotinylated antibody and staining reagents. Stimulated cells become very sticky and their complete removal may require incubation with an enzymatic cell-detachment solution. Some protocols recommend up to 0.05% Tween-20 in the wash buffer. A drawback of using Tween-20 is that it sometimes reduces the hydrophobicity of the PVDF membrane to such an extent that the PVDF membrane leaks, completely ruining the experiment.
Washing can be performed in several ways: manually (multichannel pipette or squirt bottle) or (semi)automatically using a multichannel washer or an ELISA microplate autowasher.
Since damaging of the membrane is a serious risk when using an automatic plate washer and the fact that both sides of the membrane need to be washed, a squirt bottle is the best choice for effective washing of membrane-bottomed plates. On the other hand, an automatic plate washer is highly convenient and effective for polystyrene-bottomed plates. It should be realized however, that the use of an automatic washing device may cause problems in case the probes are placed too far or too close to the bottom of the wells generating a poor wash or well-washed concentric areas in the center of the well with clearly defined spots. Automated washers also require consistent decontamination and cleaning procedures, otherwise accumulation of debris in the probes will create high and irregular background staining and slow flow.
After completing the wash, tap the plates on a dry paper towel.
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Is there an explanation for the occurrence of concentric areas lacking spots?
This phenomenon is typically caused by foam formation during washing of the plates. Particularly squirt bottles with a narrow spout produce excessive foam preventing an effective and uniform wash. The use of an automatic washing device or squirt bottle with a wide spout is usually sufficient to overcome this problem.
Picture:
Example of areas in the well lacking spots due to foam formation
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Is it possible to stack ELISPOT plates in the incubator?
It is critical that the plate is equally heated during incubation. Stacking of plates will lead to variation in temperature of the individual wells and consequently the size and frequency of spots will vary.
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How to dry the plate after color development?
Both polystyrene-bottomed and PVDF membrane-bottomed plates should be dried without a lid upside down at 37°C. When dry (after several hours), PVDF membrane-bottomed plates with enzymatically colored spots should be stored in the dark at a dry place at room temperature to prevent bleaching of spots. Storage of plates with silver-stained spots do not require special storage conditions.
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My question is not mentioned here, how can I contact Aniara?
For more information or questions regarding the ELISPOT assay or other U-CyTech products please contact us.
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