Frequently asked questions ELISPOT and FluoroSpot assay

The enzyme-linked immunospot (ELISPOT) assay is a widely used method for monitoring immune responses. The assay is a highly sensitive method for the ex vivo quantification of cytokine or antibody secreting cells after stimulation with an appropriate stimulus in vitro (Cox et al. 2006). In general, each cell can be detected by ELISPOT if a characteristic protein is released and specific high affinity antibodies recognizing the protein are available.

The ELISPOT assay is comparable to an enzyme-linked immunosorbent assay (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.
First of all, coating antibodies, specific for the protein of interest, are immobilized onto a solid phase (96-well plate). Immune cells (e.g. PBMC or activated B cells) are subsequently added in the wells and incubated in the presence or absence of stimuli that activate cytokine or antibody secretion. The released proteins are trapped by the coated antibody in the area directly surrounding the producing cells. After washing the wells to remove the immune cells, any cytokine or antibody that was captured by the coating antibody remains in the well. Captured proteins are then visualized by a combination of a second specific detection antibody, a conjugate and a precipitating substrate.
A single cell forms a colored 'footprint' (spot) on the bottom of the well representing its secretory activity. The frequency of spot forming cells can be quantified 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 or antibody secreting cells in the ELISPOT assay.

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The dual color FluoroSpot assay is a modification of the T cell ELISPOT assay. It is developed for simultaneous detection of two cytokines released by a single T cell. The assay is based on the use of fluorescent conjugates, which can be visualized by fluorescence microscopy or by an ELISPOT reader equipped with a fluorescent light source.
Two different coating antibodies, specific for the cytokines of interest, are immobilized on a solid phase (96-well FluoroSpot plate). Immune cells (e.g. PBMC) are incubated in the presence or absence of stimuli that activate cytokine secretion. After binding of the released cytokines, the cells are washed away. Areas where cytokines are captured by the two coating antibodies are detected by incubation with a mixture of two different anti-cytokine detection antibodies, followed by an incubation step with two fluorescent labeled conjugates.
The generation of spots that emit light at different wavelengths reveals the precise location of single and double cytokine-producing cells.

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ELISPOT assays are one of the most sensitive and specific techniques available for monitoring T and B cell responses permitting the ex vivo identification of cells actively secreting signaling proteins or antibodies. In addition the assay is relatively fast, highly efficient and the limit of detection achieved 1 in 100,000 cells.
Major advantages of the ELISPOT assay are its relatively easy performance and its potential for high throughput screening. Because of the short-term in vitro culture, the measured response closely mirrors the in vivo T or B cell frequency of an individual.

The T cell ELISPOT assay has the lowest detection threshold among the assays available for detection of T cell responses. Classical T cell assays are Mixed lymphocyte reaction (MLR) and Cytotoxic T lymphocyte (CTL) assays that measure CD4⁺ or CD8⁺ T cell mediated immune responses. Both MLR and CTL assays have their drawbacks including the use of radioactivity, low throughput screening, decreased sensitivity in cryopreserved specimens and technical burden. A more modern test, RT-PCR analysis, is a sensitive assay to measure T cell responses. However, this assay detects mRNA instead of secreted protein.

The B cell ELISPOT assay is the assay of choice to identify and determine the number of antibody secreting cells (ASC) and quantitate the frequency of circulating memory B cells. The traditional method to monitor a B cell response generated after immunization or infection is to measure specific antibody titers in serum by ELISA. However, antibody levels in serum do not provide information on the number of ASC, the memory B cell response and the status of the immune response such as non-responsiveness to vaccines. Vaccination normally induces long-term or even life-long protection mediated by the presence of circulating antigen specific memory B cells that do not spontaneously produce antibodies ex vivo. Therefore, memory B cells require antigenic stimulation to differentiate into ASCs, what can be performed in the B cell ELISPOT assay.

In the sensitive FluoroSpot assay two cytokines can be detected in one assay. Therefore, fewer cells are required.

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An ELISA determines the total concentration of the secreted signaling protein or antibody, whereas an ELISPOT/FluoroSpot assay detects individual cytokine or antibody secreting cells answering the question 'what is the frequency of secreting cells?'. Therefore, ELISPOT/FluoroSpot assays should be used not 'instead of ' but rather 'in addition to' ELISA.
An ELISPOT/FluoroSpot assay can be 100 to 400 times more sensitive than a conventional ELISA, because the secreted protein is captured directly onto the well of an ELISPOT/FluoroSpot plate before it will be diluted in the culture supernatant, degraded by proteases, or captured by receptors on adjacent cells.

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The ELISPOT/FluoroSpot 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. For more information, please see ELISPOT applied in research.

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The most important information that can be achieved from ELISPOT/FluoroSpot assays is the frequency of antigen-specific T or B cells within a pool of different cell types. This frequency reflects the extent of the cellular of humoral immune response.

Pictures:
Example of human IgG B cell ELISPOT results (enzymatic staining):
Used in the assay: 1x10⁵ human PBMC/well
PBMC of an individual vaccinated with tetanus toxiod more than 15 year ago.
Memory B cells were activated with IL-2 and R848 during an in vitro preincubation step.
Upper: Number of tetanus toxoid specific IgG secreting cells.
Lower: Total number of IgG secreting cells.

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Whole blood should be kept at room temperature (RT; 20-26 °C) 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 8 h after blood draw. For the T cell ELISPOT and FluoroSpot assay, blood samples can be gently mixed on a tube roller until PBMC isolated.

The recommended anti-coagulants are heparin and citrate. For both substances there are no reported adverse effects on cellular function in the ELISPOT and FluoroSpot assay.
EDTA, on the other hand, inhibits coagulation through calcium chelation which impair cytokine induction during antigen-specific re-stimulation and is therefore dissuaded as anti-coagulant for blood samples to be used in the T cell ELISPOT/FluoroSpot assay.

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PBMC are isolated by density gradient centrifugation (using e.g. Ficoll). Since red blood cells will interfere with the ELISPOT assay it is not recommended to work with whole blood.

Monocytes are critical as antigen-presenting cells (APCs) for T cells and too low numbers of monocytes can lead to lower spot forming cells in the T cell ELISPOT/FluoroSpot assay. Particularly cryopreserved samples may contain a decreased number of monocytes. Replenishment of monocytes may therefore be necessary.

The presence of dead cells in PBMC preparations can impact the functioning of both the T and B cell ELISPOT assay and the FluoroSpot assay, and a cut-off of ≥ 80% viability is recommended as a threshold. In some cases, even though the number of dead cells is low, there may be a lower number of spots because of apototic cells present during the isolation procedure or during culturing.

Pictures:
Example of human IFN-γ T cell ELISPOT results (silver staining):
2x10⁵ PBMC/well
Upper: monocyte depleted sample
Lower: normal sample

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ELISPOT and FluoroSpot sensitivity critically depends on cell density. For example, in the T cell ELISPOT assay a high number of cells enhances the probability of contact between stimulating antigen-presenting cells and responding T cells. Is the number of cells too low than cell contact is inadequate for optimal responses. However, too many cells can lead to overlap of individual spots and high background staining (50-250 spots/well is optimal for spot counting).

In a 96-well ELISPOT/FluoroSpot plate, a maximum of 3x10⁵ cells can be added to each well for antigen-specific responses. This number of cells forms a tight monolayer on the bottom of the wells. Higher numbers of cells will lead to pilling up of cells and linearity between cell input and detected spot frequency is lost. In general, 2x10⁵ cells/well result in less than 100 antigen-specific spot forming cells per well.

For polyclonal stimulation in the T cell ELISPOT/FluoroSpot assay (such as mitogens or PMA/ionomycin) and the determination of total immunoglobulin secreting cells in the B cell ELISPOT assay, 10-100-fold less cells (2x10² – 1x10⁵ cells/well) should be plated to obtain a spot frequency of 50-100 spot forming cells per well. 

Pictures:
Example of Human IgG B cell ELISPOT results (enzymatic staining):

Memory B cells were activated with IL-2 and R848 during an in vitro preincubation step.
Shown: Total number of IgG secreting cells.

Upper: 5x10³ human PBMC/well
Lower: 5x10⁴ human PBMC/well (resulting in too many spots to count)

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T cell ELISPOT assay:

A 24-42 h preincubation step at high cell density (>10⁶ cells/well/ml) can be required when full-length proteins or long peptides are used as stimulus in the ELISPOT assay. 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. The high number of cells enhances the probability of contact between stimulating and responding cells. Omitting this step may results to a significant lower frequency of spot forming cells. On the other hand, small (synthetic) peptides (8-12 mer) can directly be presented by APC to CD8⁺ cells and consequently need no preincubation step.

A preincubation step can also be useful when the analyte of interest requires a longer incubation.

Detailed information can be found at Cell sample preparation T cell ELISPOT assay.

Example of IL-2 specific spots produced by mouse (C57bl/6) splenocytes using the Mouse IL-2 T cell ELISPOT kit (CT435-PR):

The recommended cell culture medium is RPMI-1640 supplemented with 2 mM L-Glutamine, 100 units/ml penicillin, 100 μg/ml streptomycin and 10% fetal calf serum (FCS).

It should be mentioned that some batches of FCS may non-specifically activate the cells and can therefore be the cause of background spot formation. Different batches of FCS should therefore be pretested on both low background reactivity as well as high antigen-specific responses before using it in the ELISPOT/FluoroSpot assay.

In the T cell ELISPOT assay human, non-human primate or rodent serum can be used as alternative for FCS. This serum should selected on low background reactivity/high antigen-specific responses as well. When the T cell ELISPOT assay is performed without preincubation step, serum-free medium (AIM-V) supplemented with 100 units/ml penicillin and 100 μg/ml streptomycin can also be a good alternative. 

Do not use human, non-human primate or rodent serum as growth supplement in the B cell ELISPOT assay. The antibodies in the serum will interfere with spot formation.

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Appropriate controls are important for the validation of the ELISPOT and FluoroSpot assay. Choice of controls depends on the cells of interest and type of experiment.

Positive control in the T cell ELISPOT and FluoroSpot assay
- Cell are activated with antigen-specific or polyclonal stimuli.
- High number of spots indicates that the assay works properly.

Both antigen-specific and polyclonal stimuli can be used. However, antigen-specific stimuli are preferred since cells of various species respond differently to polyclonal stimuli. For the human system, vaccine proteins (tetanus toxoid, hepatitis B, etc.) or a pool of synthetic peptides of common human viral epitopes are excellent positive controls. When using vaccine proteins, it should of course be known whether individuals have been vaccinated.

Some animal models are skewed to a Th1- or Th2-like response. This can result in a low number of spots in the positive control. Therefore, please check if your animal model is expected to be able to produce the cytokine of interest.

Negative control in the T cell ELISPOT and FluoroSpot assay
- Cells are incubated without any stimuli.
- No spots or spontaneously occurring spots appear

Spontaneously secreting cells: if these cells are not triggered by e.g. culture medium ingredients (false positive results that should be avoided), they may be caused by an activated immune system of the donor without us realizing it. The number of spontaneously occurring spots may vary from donor to donor and at different time points for a donor within the observation period.

Background control in the T cell ELISPOT and FluoroSpot assay
- Run the assay without adding cells to the well of the plate.
- No spots or reagents/cell culture media produce false spot-like structures.

Positive control in the B cell ELISPOT assay (memory B cells only)
- Cells are incubated in wells coated with an antibody to species-specific immunoglobulin.
- High number of spots provides information whether the assay works properly and provides data to determine the ratio of antigen-specific versus total antibody secreting cells.

Background control in the B cell ELISPOT assay
- Cells are incubated in wells not coated with antigen or antibody.
- No spots or reagents/cell culture media produce false spot-like structures.

To: An overview of general cell stimuli commonly used in ELISPOT and FluoroSpot assays

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In case cells in the PVDF membrane-bottomed ELISPOT plate are contaminated with infectious agents such as enveloped viruses (e.g. HIV, SIV), the wells can be decontaminated after the preferred cell incubation time by first removing the cells from the well, followed by washing 5 times with PBS, adding 300 µl 4% paraformaldehyde (in PBS) and then incubate max. 5 minutes at room temperature (20-26 °C). 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 in the T cell ELISPOT assay.

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Selection of optimal primary and secondary antibodies in an ELISA system does not imply that these are also the most optimal antibodies for an ELISPOT assay. The ELISPOT antibody pairs are selected by the manufacturer on basis of extensive optimalization and validation and should be used as a matched antibody pair for the ELISPOT assay.

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For optimal results, the coating antibodies should stay in the wells of the ELISPOT/FluoroSpot plate for minimal 16 h at 4 °C. The blocking buffer can be incubated for one to several hours at room temperature (RT; 20-26 °C) or overnight at 4°C without any problem. Incubate the detection antibodies 2 h at RT or overnight at 4 °C (incubate the detection antibodies of the FluoroSpot assay protected from light). The conjugate can be incubated (protected from light) for 1 h at RT or overnight at 4 °C.

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Antibody coated plates can be prepared 7 days ahead. Seal the ELISPOT plate against evaporation and store the plate 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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Our ELISPOT kits make use of an enzyme, horseradish peroxidase (HRP), for visualization of the protein secreted by the cells.
The advantage of using HRP as conjugate is its rapid turnover rate (spots develop rapidly). The HRP substrate AEC (3'-amino-9-ethylcarbazole) forms intense red colored spots, but may bleach in short period of time when stored in daylight. Therefore, plates should be protected from light and moisture during storage at room temperature (20-26 °C). 
The spots can be evaluated by a reflected light microscope or an automated ELISPOT reader.

Picture:
Example of Human IFN-γ T cell ELISPOT
2x10⁵ PBMC/well; stimulus: viral peptide pool
Using streptavidin-HRP conjugate and AEC substrate. 

Our FluoroSpot kits use a fluorescent labeled conjugate containing Alexa Fluor 488-labeled anti-FITC antibodies and R-PE-labeled streptavidin. A spot enhancer is used to enhance the fluorescence signal.
A fluorescent microscope or automated ELISPOT reader with fluorescent applications is required to visualize the spots.
Fluorophores lose their fluorescence due to damage caused by light (photobleaching). Therefore, plates should be protected from light and moisture during storage at room temperature (20-26 °C).

Picture:
Example of Monkey IFN-γ / IL-10 FluoroSpot
1x10⁴ PBMC/well (rhesus macaque); stimulus: PMA/ionomycin
Using Alexa Fluor 488-labeled anti-FITC antibodies and R-PE-labeled streptavidin conjugate.
In green: IFN-γ. In red: IL-10. In yellow: IFN-γ/IL-10.

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Each spot within an ELISPOT/FluoroSpot well is the 'footprint' of a single cell that has released a relatively high amount of cytokines or antibodies. True spots have a dense center with a light outer ring caused by the diffusion of the secreted protein from the producing cell.
The color depth or the size of spots depends on the amount of secreted proteins. Only strong and well-defined spots should be counted; any small or faint spot is likely to be an artifact and should be ignored.

Sometimes small and very dark 'spots' appear in the background that are not generated by secreted cytokines or antibodies. 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 in the T cell ELISPOT they may originate from small cell membrane fragments firmly attached to the solid support carrying receptor bound cytokines.
Artifactual spots can also be caused by the aggregation of antibodies used in the process of coating and detection, incomplete removal of cells from the plate after incubation, DNA precipitates or simply dirt. The use of other samples than PBMC, like cell lysates or minced tissue, can cause artifactual spots as well.

If possible, it is important to exclude (or prevent the forming of) artifacts and small background spot before visual evaluation of the ELISPOT/FluoroSpot plate. Thoroughly washing of the plate between all incubation steps and after completion of the coloring reaction minimizes false spot formation. Dust particles can be removed by blowing 4-5 bar compressed air into the well.

The average size of a true spot is 5,000-10,000 square microns, 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 protein-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/FluoroSpot data and offers various procedures to overcome variable background intensity problems and to distinguish true spots from artifacts by image analysis.

In ELISPOT/FluoroSpot assays, a wide spectrum of spot sizes and densities can be seen. In summary, when evaluating results, cut-off values for the different analyses need to be set for the minimum spot size to be counted and the maximum spot size has to be defined to identify clusters of cells. The minimum and maximum 'gates' set will critically affect the number of spots counted. For this reason, one of the main goals of image analysis has been to establish absolute criteria for gating for each different ELISPOT/FluoroSpot assay.

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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. In addition, the movement of plates during cell incubation may cause the cells to roll generating irregular or oblong-shaped spots.

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Dust particles can be present in the wells. Upon visual evaluation, these 'spots' vary in size, are highly irregular and usually black. By blowing 4-5 bar compressed air into the well, this problem can easily be solved.

See also 'What are the characteristics of a spot?'.

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Our ELISPOT kits are suited for the use with polyvinylidene fluoride (PVDF) membrane-bottomed plates. 96-well MultiScreen_HTS Immobilon-P (IP) plates from Millipore (e.g. cat. no. MSIP S4510) are recommended.

Our FluoroSpot kits are provided with PVDF membrane-bottomed plates (96-well IP-FL plates from Millipore). The PVDF membrane of these plates is smoother than the plates suitable for ELISPOT assays, resulting in less auto-fluorescence. It is not recommended to use plates suitable for ELISPOT analysis in the FluoroSpot assay.

When using hydrophobic PVDF membrane-bottomed plates a prewetting step with 70% ethanol is required. The treatment with ethanol makes the membrane hydrophilic and ensures optimal binding of the coating antibody resulting in better sensitivity (increased spot number) and more accurate quantitation (more sharply defined spots) (Weiss A.J. 2012). After maximum 1 minute of prewetting with 25 µl 70% ethanol, the membrane should be rinsed thoroughly by adding coating buffer to the well to efficiently wash out the ethanol before the coating antibody is added to the wells. Once the membrane is ethanol-treated, it must be kept wet during the entire assay procedure. Overtreatment with large volumes of ethanol, more concentrated ethanol and longer exposure time can lead to trapping of residual liquid between the membrane and underdrain, which may result in poor assay performance. 

There are both sterile (Millipore cat. no. MSIP S4510 and MSIP S4W10) and non-sterile (Millipore cat. no. MSIP N4510, MSIP N4550, MSIP N4W50 and M8IP S4510) 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 safely used without contamination problems.

Using clear MultiScreen_HTS IP plates (Millipore cat. no. MSIP S4510, MSIP N4510, MSIP N4550 and M8IP S4510) or white MultiScreen_HTS IP plates (Millipore cat. no. MSIP S4W10 and MSIP N4W50) with a PVDF membrane bottom is whatever the user prefers best. Both type of plates will give a different appearance in the automated ELISPOT reader. This should not affect the evaluation of the plates. However, the automated ELISPOT reader may find it more difficult to locate the center of the well of white plates due to reflections of the wall.

Our 2-plate T cell ELISPOT kits are supplied with 2 PVDF membrane-bottomed plate from Millipore (cat. no. MSIP S4510).

We advice to remove the underdrain of the plate after the detection antibody incubation step and to wash both sides of the PVDF membrane in each wash step.

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It is important to use tips with a wide opening for the pipetting of cells to avoid shearing stress upon the cells that may induce apoptosis and thus inhibition of spot formation. Make sure the cells and other liquids are added smoothly. Adding the cells fast/abruptly or adding any liquids after adding cells into the well may lead to an uneven distribution of spots.

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A critical issue in ELISPOT/FluoroSpot analyses, is the washing procedure. Washing of ELISPOT/FluoroSpot plates is necessary to remove cells and residual biotinylated antibody and staining reagents. 
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 with a wide spout is the best choice for effective washing of membrane-bottomed plates. The bottle should be used to fully fill the wells with wash buffer. Subsequently wells are emptied by a firm ‘shake-out’ action. Repeat this as many times as mentioned in the manual.
After completing washing, tap the plates on a dry paper towel.

We advice to remove the underdrain of the plate after the detection antibody incubation step and to wash both sides of the PVDF membrane in each wash step thereafter.

See also 'Direction for washing ELISPOT and FluoroSpot plates'. 

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Stacking of plates may lead to variation in temperature of the individual wells and consequently the size and frequency of spots will vary. Therefore, stacking plates is not recommended.

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PVDF membrane-bottomed plates should be dried protected from light, without a lid and upside down at room temperature (RT; 20-26 °C). It is important that the membranes are dried completely before evaluating the plates. When dry, PVDF membrane-bottomed plates should be stored protected from light at a dry place at RT to prevent bleaching of spots.

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Spots from the FluoroSpot assay suffer from photobleaching. When light of the appropriate wavelength is focused on a fluorophore, it transitions from the ground state to the excited stage. In its excited state, it may interact with other molecules and undergo permanent covalent modifications. Therefore, all fluorophores will eventually fade. When the plates are stored protected from light in a dry place at room temperature (20-26 °C), the spots are quite stable (several weeks to months). However, it is recommended to evaluate the FluoroSpot plate within a week of completion of the assay. Please, keep in mind that repeated expose during evaluation (for example, while setting up an automated reader) will also lead to photobleaching of spots.

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For PVDF membrane-bottomed ELISPOT plates a reflected light microscope or an automated ELISPOT reader is needed for spot counting.

To evaluate our FluoroSpot plates a fluorescent microscope or an automated ELISPOT reader equipped with a multicolor optical filter set for FITC (Excitation/ Emission: 495/519 nm; visualizing green spots) and R-Phycoerythrin (R-PE, Excitation/ Emission: 546/578 nm visualizing red spots) is needed.

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The researchers of U-CyTech build on >20 years experience in the development and performance of ELISPOT assays for human, monkey, mouse and rat species. They are more than happy to help you with additional advice and to share their experiences.

Please contract our customer service:
E-mail: cs @ ucytech.com

U-CyTech biosciences
Yalelaan 48
3584 CM Utrecht
The Netherlands

Phone: +31.85 073 1460

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