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The key to consistency in an ELISpot assay is to use stable cell cultures and develop good cell handling techniques. Considerations must be made on the source, preparation, counting, and viability of cells used in ELISpot assays. Please click on one of the topics below for a discussion of ELISpot cell choice and handling considerations and best practices.
Considerations must be made on the source, preparation, counting, and viability of cells used in ELISpot assays.
Fresh vs. Frozen - PBMCs or enriched T-cell subsets constitute the bulk of ELISpot assays. Once purified from blood, PBMC can be cryopreserved and thawed without loss of functional activity. Given the often precious nature of disease samples, there are multiple benefits to cryopreservation:
(1) Data can be independently reproduced. (2) Multiple analytes can be assessed. (3) Patient samples can be stockpiled and assayed simultaneously, thus minimizing the potential inter-assay variability.
For optimal recovery of viable cells, follow these recommendations: (A) during freezing, have cells and freezing medium at room temperature prior to mixing and (B) during thawing, to minimize osmotic lysis during washing of thawed cells, transfer cells to a 15 mL conical tube on ice and slowly add cold medium.
An additional challenge with using frozen PBMCs is cell clumping during the thawing process. Clumping is often caused by the presence of free DNA and cell debris; it appears to be related to both the donor source and blood handling.
In particular, clumping occurs more frequently when blood has been stored overnight prior to PBMC isolation. Spot count results for overnight-stored blood showed a dramatic decrease when compared with responses for the corresponding PBMCs isolated from fresh blood.
The greatest decreases in signal were detected for samples in which the highest degree of cell clumping was observed. To improve assay performance, we recommend addition of Benzonase® nuclease, which degrades all forms of DNA and RNA, to the assay medium for the first two wash steps during the thawing procedure. The results from overnight blood PBMCs processed with Benzonase® nuclease more closely approximate the results obtained with cells isolated from fresh blood. Moreover, Benzonase® nuclease addition resulted in no changes to cell viability or changes in the expression of certain surface markers, including CD4, CD8, CD38, or CD62L.
Cell counting and the value of percent viability – Once Ab steps have been standardized, differences in quantified cell yield and integrity of each sample presents the greatest source of assay variability. While total cell counts are important, a more critical factor to consider when setting up the culturing component is cell viability.
Determining the percentage of dead and apoptotic cells is not only important for culture setup, it also provides quantitative information on the overall quality of the sample. The latter component is particularly useful when assessing success/failure of the freeze-thaw process. Manual counting methods, such as Trypan Blue exclusion using a hemacytometer, lack accuracy due to user subjectivity. Further, these methods do not provide a measurement of the apoptotic fraction. Automated cell counting via flow cytometry using fluorescent dyes, such as Merck’s Muse™ cell analyzer and Count & Viability assay, demonstrated superior precision to manual methods for the enumeration of viable cells.
Cells per well and replicates – On average, T-cell ELISpot counts show linearity for PBMCs in the range of 100,000-800,000 cells. Where possible, cells should be serially diluted and plated in triplicate. Unfortunately, given the restrictions of well size in 96-well plates (0.3 cm2), seeding more than 400,000 cells per well may result in overcrowding and cell stacking.
The consequence here is creation of diffuse spots due to indirect contact of the cells with the Ab-coated membrane. To best monitor instances where the frequency of Ag-specific responders is very low, and higher cell loads are required, either perform assays in larger wells or perform replicate wells at maximal cell density. By using replicate wells, spot counts from all the wells can be summed to derive the response frequency (SFU/total cells seeded). During incubation, we do not recommend plate-stacking, as this can lead to variations in temperature between the plates and potentially differences in spot size and/ or number. Also, it is important that plates are subject to minimal agitation, because movement can lead to localized cytokine diffusion and loss of spot sharpness.