Step-by-step guide to FluoroSpot

FluoroSpot is the multiplex version of ELISpot. The assay combines the sensitivity of ELISpot with the capacity to study secretion of several analytes simultaneously. This is an in-depth guide for each step of the FluoroSpot protocol, including tips and tricks, key details to think about, and best practice.

FluoroSpot is an immunoassay used to quantify analyte-secreting cells. Just like in the ELISpot assay, target proteins secreted by cells are captured by specific antibodies immediately after secretion and throughout the stimulation process, making it an extremely sensitive sandwich assay. The difference to ELISpot lies in the mode of detection: where ELISpot relies on an enzymatic reaction, FluoroSpot utilizes fluorescence. This opens up for multiplex analyses of several analytes simultaneously, enabling studies of cell populations with different functional profiles.

We developed the technique in 2008 and have continually launched new kits and refined the protocol. Thus, we can offer many tips to fine-tune and maximize the success of your assay. We will discuss such suggestions in this guide, starting where the assay begins – with coating the FluoroSpot plate – and continue through the steps of the assay.

The general steps of a FluoroSpot assay

Ethanol pre-treatment and plate coating – be meticulous

The PVDF membrane can only bind the required amount of capture antibody after ethanol pre-treatment which makes the membrane hydrophilic. In addition, the spot appearance is optimized by using the recommended concentration of capture antibody (this might differ depending on analyte, but is usually 15 µg/ml). If you dilute the capture antibody to lower than recommended concentrations, you risk getting fuzzy spots that are hard to count.

Handle the plate carefully

The following tips are applicable for all steps of the assay:

• Do not allow the plate membrane to dry out during the assay. If your plate membrane does dry out during ethanol pre-treatment – don’t panic, simply repeat the ethanol pre-treatment steps.

• Wash plates using a multichannel pipette when you need to work in a sterile environment, e.g., in a laminar flow hood. When sterile conditions are not required (please check the protocol of the product), you can use an ELISA plate washer but make sure to adapt the washer head position to the shallower wells of FluoroSpot plates.

• Do not use Tween 20 in your wash buffer. We know it’s commonly included when running ELISA assays, but please don’t include it for FluoroSpot. Tween 20 may damage the PVDF membrane and/or solubilize cell membranes, both of which will negatively affect spot formation and appearance.

• If you need to dissolve your antigen of interest in DMSO, make sure that the DMSO in the cell culture is diluted to a working concentration of below 0.5%. DMSO at a higher concentration may damage the PVDF membrane as well as the cells, and cause artifacts.

• Avoid splashes when emptying plates, as liquid might splash back into the wells. As a final emptying step, tap the plate gently against a paper towel.

Whether you coat the plates yourself or run pre-coated plates from us, by this stage the FluoroSpot plate will be ready for your cells (after blocking the plate by adding 200 μl of the same medium as will be used for your cells, and incubating at room temperature for 30 min – follow the protocol). Being a cell-based assay, cell-viability is key to a successful FluoroSpot assay.

Cells are incubated in the presence or absence of an activating stimulus (an antigen or polyclonal stimulatory agent) at an appropriate temperature (usually 37 °C). The incubation period should allow for optimal analyte secretion and thus can last from a few hours up to a few days. We recommend an assay setup with triplicate wells for all conditions.

Most cell sources can be used – as long as the cells are alive

FluoroSpot can be applied to basically any cells to investigate protein secretion. We have seen publications with a wide range of cells, including human blood cells, mouse spleen cells, cell cultures, cells from mucosal tissue, heterogeneous cell populations and purified homogeneous cells, and cell clones. The most commonly used cells, at least from human samples, are peripheral blood mononuclear cells (PBMCs). They can be obtained from blood by density centrifugation using e.g., Ficoll. The resulting PBMCs can be used directly or may be frozen for later use.

Using samples with a high proportion of viable cells and a low proportion of apoptotic or dead cells will give you the greatest likelihood of success. This is not unique for FluoroSpot, but true for any cell-based assay. If your sample contains a high proportion of dead cells, you cannot rely on your final results as they are based on the ratio of responsive cells (spot-forming units) to total cells.

Freezing and thawing cells is not a problem if you do it right. But an important note is that PBMCs should be prepared as soon as possible after blood collection. This is because cells prepared from blood samples older than around 8 hours may be contaminated with activated granulocytes which can interfere with the results. If blood samples must be stored before preparation (for example over-night if you received the sample late in the afternoon), high-quality PBMCs can be obtained by:

• Gently agitating the blood samples
• Diluting the blood samples 1:1 in PBS or RPMI
• Depleting the granulocytes from the blood samples using commercial depletion kits, e.g., from Stemcell Technologies

Choose a cell culture medium appropriate for your cells

The cell culture medium plays a crucial role in the well-being of your cells. We recommend RPMI 1640 with L-glutamine and 10% fetal calf serum (FCS) for most cells. At Mabtech, we usually supplement the medium with HEPES to maintain the pH of the cell culture and antibiotics to decrease the risk of contamination. Since RPMI 1640 might not be the optimal medium for your experiment, you must do the homework of checking which medium is best for the cells you are studying.

If FCS cannot be used in your assay, it is possible to use a serum-free medium containing defined proteins as a substitute for serum. Of all serum-free media we have tested, AIM-V works best for FluoroSpot.

Regardless of which cell culture medium you choose, it’s essential to use the same medium throughout the assay, including the blocking/conditioning step before adding the cells. Using the same media ensures the cells' environment remains constant in each step, reducing the number of variables that could affect the final results through non-specific stimulation. 

Choose a suitable cell number

At this point, the plate has been coated with capture antibody and conditioned with cell culture medium for at least 1 hour at room temperature. Before adding cells to the plate, you need to count and adjust them to the required concentration in cell culture medium.

Cell counting can be done with an automated cell counter or trypan blue staining using a microscope. Because only living cells will be able to secrete the analyte, make sure to exclude dead and, if possible, apoptotic cells from the cell count. If the cell concentration turns out to be lower than required, you will need to concentrate the cells by centrifugation.

The following should be considered:

• If unsure, start with 250,000 cells per well
  The number of cells in each well must be adapted to the cell type and the expected frequency of secreting cells: The lower the expected frequency of responding cells, the higher the number of cells per well. If the expected frequency is 1 in 10,000 cells, you will need approximately 250,000 cells per well. Conversely, when the frequency is high, for example 1 in 100 cells, it’s sufficient to seed 50,000 cells per well. In general, antigen-specific responses require higher cell numbers (e.g., 250,0000 cells per well) than polyclonal/mitogenic positive controls (e.g., PHA, 50,000 cells per well).

• Cells “like” to be in contact with other cells
  The number of spot-forming units measured in FluoroSpot does not always correlate with the total number of cells because a minimum number of cells is required for proper cell-to-cell contact and thus optimal stimulation. For example, when analyzing antigen-specific T cells in a PBMC sample, total cell numbers should not be less than 200,000 cells per well.

• Avoid vortexing
  Resuspend cells and pellets gently using a pipette.

• It is sometimes beneficial to stimulate cells before adding them to the FluoroSpot plate
  For analytes that need a longer time for secretion to be initiated, it is possible to pre-stimulate the cells (preferably in round-bottom polypropylene tubes or plates to augment cell contact) before adding them to the FluoroSpot plate. This is good practice in certain circumstances, e.g., when stimulating memory B cells for 72 hours with R848 + IL-2 to facilitate differentiation into immunoglobulin-secreting plasma cells. If you choose pre-stimulation, it is important to note that cells must be washed before addition to the FluoroSpot plate to avoid background staining of the membrane.

Seed and stimulate the cells

When you have chosen the cell number that is appropriate for the experiment, it’s finally time to seed the cells onto the plate and stimulate them. In addition to your stimulus of interest, we recommend you to include three control conditions:

Choosing the relevant compounds for control stimulation is largely dependent upon the cell type and the nature of your experiment. Stimuli can be monoclonal antibodies (anti-CD3 for T cells) or peptides (CEF peptide pool for CD8+ T cells, CEFTA peptide pool for CD4+ T cells), synthetic compounds (R848+IL-2 for B cells) or substances purified from bacteria or plants (PHA for T cells). Antigen-specific stimulation using peptide pools or monoclonal antibodies are preferable in our view, since these reagents are well defined. More on peptide pools and polyclonal stimuli here.

For all types of stimuli, antigen-specific or polyclonal, we recommend either of the following two techniques:

If your samples take up only a part of the plate, fill the surrounding wells with medium only (100 µl per well) to minimize evaporation from your samples during incubation.

Put the plate in the incubator

With the cells and stimuli added to the plate, the next step is to put the plate into an incubator. In general, the temperature of the incubator should be 37 °C with a CO₂ level of 5%. Two things to think about when incubating cells:

• Wrap the plate in aluminum foil
  Most incubators maintain an optimal level of humidity, but it is good practice to wrap the plate in aluminum foil during incubation to reduce the risk of evaporation of the cell culture medium.

• Place the plates next to – not on top of – each other
  Do not stack plates in the incubator. Stacking plates can lead to uneven conditions between the plates (e.g., temperature, CO₂ level, evaporation rate), which may affect the cells and thereby the spot numbers.

FluoroSpot uses specific capture antibodies in the bottom of the well to capture cytokines, immunoglobulins, or other target proteins immediately after secretion and throughout the stimulation process. This is what makes FluoroSpot so sensitive.

With the plate in the incubator, you can now just wait for the antibodies to capture the analytes. It is important to note that:

• Different analytes have different kinetics
  Consider the kinetics of secretion of the protein when establishing the optimal cell incubation time. Some analytes, such as granzyme B, are fully secreted only after more than 48 hours of stimulation, whereas others, such as IFN-γ, can be analyzed after only 12 hours. If you are studying both analytes, choose the longer incubation time. It will not have any negative effect on the “quicker” analyte.

• The plate must not be moved during incubation
  Avoid moving the plates during the incubation, as this can negatively affect spot formation. Even small movements should be avoided, so close the incubator door gently.

Capture effects and co-stimulation

When capture antibodies with different specificities are coated together, capture of one cytokine may affect the secretion of other cytokines, a phenomenon called “capture effects” or “cytokine absorption effects”. For example, presence of IL-2 capture antibodies may result in reduced activation of T cells, as capturing of IL-2 decreases the amount of IL-2 available for the cells. Thus, downstream IL-2-dependent cytokine secretion may be dampened. Luckily, capture effects can often be counteracted by addition of an anti-CD28 mAb, providing a co-stimulatory signal to T cells and thereby restoring cytokine secretion (see example in figure below).

Depending on cells and stimuli used, optimization of anti-CD28 concentration may be necessary, since too high concentration can lead to elevated non-specific cytokine secretion. Also note that capture effects are more pronounced in positive controls than in antigen-specific responses.

Our FluoroSpot Plus kits are evaluated for capture effects, and in most studies of T cell responses we recommend co-stimulation with anti-CD28. With FluoroSpot Flex, however, it is possible to combine analytes as you wish to build your own kit. Most analyte combinations work just fine, but there are a few combinations that we advise you not to use. For guidance look at our analyte combination table.

If your specific analyte combination isn’t in the analyte combination table, and you are unsure, you can investigate capture effects like this:

1. Coat wells with single capture antibodies, one set of wells per capture antibody.

2. Coat wells with a mixture of the capture antibodies.

3. Compare the number of spot-forming units in the different single conditions to the mixture condition. If you see a difference between the single coated wells and the mixture wells, you might have identified a capture effect.

4. Assess the compensatory effect of anti-CD28 mAb by comparing cells cultured with and without the anti-CD28 mAb in the same setup as steps 1-3 above.

Capture effects and its countermeasures can be perceived as a bit artificial, but it’s no different than e.g., blocking the Golgi apparatus when doing intracellular cytokine staining in flow cytometry. All in vitro assays are models of reality. It’s just about finding a setup that suits your scientific question, allowing you to detect differences between sample and control.

After the incubation step, the cells have secreted analytes in response to the stimulus, and the analytes have been captured by the specific antibodies coated on the plate. Time to detect. It might be obvious, but we’re saying it anyway: With FluoroSpot, you are detecting the footprint of the cell that was there, secreting the analyte you are studying. Thus, begin by washing away the cells: empty the plate and wash 5 times with PBS (200 µl per well).

1. Pour some FluoroSpot enhancer in e.g, a petri dish to allow the use of a multipipette.

2. Empty the last PBS wash by flicking the plate into a waste container.

3. Using the multipipette, add FluoroSpot enhancer (50 ul per well) and leave the plate at room temperature for 5-15 minutes.

4. Remove excess FluoroSpot enhancer by flicking the plate into a waste container.

Important: Try to remove as much excess FluoroSpot enhancer as possible. In the video above, we tap the plate against paper towels. Since that recording, we have discovered that some brands of paper towels give off dust particles which can cause fluorescent artifacts. Thus, instead try to flick multiple times over the waste container.

When analyzing plates it is important to consider that, although many processes are automated in a reader, you are in control over which spots to count. With Mabtech Apex™, the software for ASTOR and IRIS, the default count settings are adequate for most situations. But, similar to gating populations in flow cytometry, you can use sliders for size and intensity to determine which spots you consider relevant.

In flow cytometry, one uses fluorescence-minus-one controls to get an idea of where to set the gate, whereas in FluoroSpot you decide on a count setting based on your experience. As FluoroSpot is so sensitive, it’s not uncommon to have spots in your negative control wells. That’s ok! We highly recommend to not subtract these spots from the “real” spots, but rather to use a statistical analysis where negative control spots are taken into account for when deciding on whether you are looking at a positive response. Please read more about this statistical method, called DFR, here.

Finally, when you have decided on a count setting, it’s good practice to choose a consistent count setting throughout the project (for all wells and plates treated in the same way).

For more information on IRIS and how to analyze plates in it, watch our video: