Cell collection and handling
for the B cell ELISPOT, T cell ELISPOT and FluoroSpot assay
Table of contents:
- PBMC derived from human and non-human primates (including cryopreservation and resting of cryopreserved cells)
- Cells from rodent origin (including cryopreservation) (ELISPOT only)
- Recommended reagents
In general, samples from humans and non-human primates are collected as peripheral blood mononuclear cells (PBMC) but may also be obtained from lymphoid organs or mucosal sites. Both fresh and cryopreserved cells can be used for ELISPOT and FluoroSpot analysis.
Blood collection and PBMC isolation
Heparinzed or citrated blood can be held at room temperature (RT; 20-26 °C) up to 8 h after collection.1-4 PBMC are isolated by density gradient centrifugation (using e.g. Ficoll) and washed twice in culture medium (8 min, 200 x g, RT). PBMC can be used directly or frozen for later use in the ELISPOT or FluoroSpot assay. Always check the viability of the cells before using in the ELISPOT or FluoroSpot procedure (especially when working with cells that have been cryopreserved); count only living cells for the ELISPOT/FluoroSpot assay.
- Specimen collection from humans and non-human primates should be carried out in accordance with CLSI document M29-A4 “Protection of laboratory workers from occupationally acquired infections”. No known test method can offer complete assurance that human- or non-human primate-derived blood or tissue samples will not transmit infection. Therefore, all human and non-human primate specimens should be considered potentially infectious.
- B cells normally constitute a minor fraction (5-12%) of PBMC. Therefore, the samples can be enriched by using e.g. negative selection by depleting non-B cells or by cell sorting.
- If blood samples have been stored >20 h, the PBMC suspensions may be contaminated with activated granulocytes. This will result in a significant reduced T cell response. Methods to prevent this contamination:
- PBMC from HTLV-1-infected human and STLV-infected monkeys may contain a high frequency of spontaneously IFN-γ producing cells.5
Cryopreservation of PBMC
PBMC can be safely frozen in culture medium supplemented with 10% DMSO and 30% to 90% FCS with a recovery of more than 80%. Alternatively, also serum-free cell freezing medium can be used for cryopreservation of cells for ELISPOT/FluoroSpot analysis (e.g. BAMBANKER).6
Freeze the cells by using a Nalgene “cryo 1 °C freezing” container or a Corning "CoolCell alcohol-free freezing" container. Cool the freezing medium and container to 0 °C before use. Suspend cells in freezing medium. The recommended cell concentration is ≥2x107 cells/ml. Insert the vials containing cells in the container and place the container overnight in a -80 °C freezer. The vials with cells are subsequently stored in liquid nitrogen.
Thawing is stressful to frozen cells. Using an appropriate and fast procedure ensures that a high proportion of cells will survive. In brief, the cells are rapidly thawed by gently swirling the cryovial in a 37 °C water bath until there is a small piece of ice left. Dropwise add 1 volume of fresh and cold culture medium containing 50% FCS (4 °C). Subsequently, 10 volumes of cold culture medium containing 10% FSC (4 °C) is added (slowly and under constant swirling) for the first wash step (8 min, 200 x g, 4 °C). Thereafter, cells are washed with fresh culture medium (RT) containing 10% FCS (8 min, 200 x g, RT).
Resting of cryopreserved PBMC (T cell ELISPOT and FluoroSpot assay only)
Several research groups have shown that detection of antigen-specific T cell responses by ELISPOT/FluoroSpot assay using cryopreserved PBMC is improved when cells are rested overnight at 37° C, 5% CO2 and 100% humidity in culture medium containing 10% FCS prior to assay (both in human as non-human primates).3,7,8,9 This resting procedure helps to eliminate apoptotic cells and allows more accurate counts of viable and functional cells. However, resting of cryopreserved PBMC does not always improve the T cell ELISPOT/FluoroSpot performance.10 Therefore, if the performance of the T cell ELISPOT or FluoroSpot assay will benefit from resting cells, depends on the internal laboratory procedure, clinical subject cohorts and the antigens used.
For rodent PBMC, the same conditions can be applied as described for human PBMC. However, most ELISPOT data so far are obtained with mouse or rat spleen cells.
Spleen cell collection and isolation
It is recommended to start isolating spleen cells within 8 h after spleen collection.
Spleens are aseptically removed from rodents and collected in culture medium. A single cell suspension is prepared by gently mechanical dissociation of small pieces of spleen tissue in culture medium through a sterile 70 µm filter (i.e. a Greiner bio-one EASYstrainer) and washed twice in culture medium (8 min, 200 x g, RT). Spleen cells can be used directly in the T cell ELISPOT assay. Always check the viability of the cells before using in the ELISPOT procedure (especially when working with cells that have been cryopreserved); count only living cells for the ELISPOT assay.
Cryopreservation of spleen cells
For the T cell ELISPOT assay is strongly recommended to work with fresh rodent spleen cells because cryopreservation of rodent spleen cells is complicated. This also applies to the B cell ELISPOT assay, but to a lesser extent. In general, cryopreservation has a negative impact on cell viability and function. By using BAMBANKER serum-free cell freezing medium, it is possible to obtain a recovery of 70-80% viable cells and cell function at levels similar to freshly isolated spleen cells. Follow the freezing and thawing procedures described in the section “Cryopreservation of PBMC”.
Gad et al. (2013) reported that resting of cryopreserved murine spleen cells before ELISPOT did not improve T cell responses.11
- BAMBANKER: Nippon Genetics Europe cat. no. BB03 (for cell cryopreservation).
- Dimethyl sulfoxide (DMSO): Sigma-Aldrich cat. no. D2650 (for PBMC cryopreservation).
- Fetal calf serum (FCS; should be selected on low background staining): Thermo Fisher Scientific cat. no. 16000.
- Ficoll-Paque PLUS: GE Healthcare cat. no. 17-1440-02 (for isolation of PBMC by density gradient centrifugation).
- PBS-I: sterile and pyrogen-free liquid PBS pH 7.4: Thermo Fisher Scientific (cat. no. 10010.
- Trypan blue solution: Sigma-Aldrich cat. no. T8154 (for cell counting and to check on cell viability).
- RPMI-1640 medium: Thermo Fisher Scientific cat. no. 52400.
- L-Glutamine (200 mM): Thermo Fisher Scientific cat. no. 25030-081.
- Penicillin-Streptomycin (100x): Thermo Fisher Scientific cat. no. 15140-122.
- Afonso G et al. (2010). Critical parameters in blood processing for T-cell assays: Validation on ELISpot and tetramer platforms. J Immunol Meth 359: 28–36.
- Bull M et al. (2007). Defining blood processing parameters for optimal detection of cryopreserved antigen-specific responses for HIV vaccine trials. J Immunol Meth 322: 57-69.
- Kierstead LS et al. (2007). Enhanced Rates and Magnitude of Immune Responses Detected against an HIV Vaccine: Effect of Using an Optimized Process for Isolating PBMC. Aids Res Hum Retrov 23: 86-92.
- McKenna KC et al. (2009). Delayed processing of blood increases the frequency of activated CD11b+ CD15+ granulocytes which inhibit T cell function. J Immunol Meth 341(1-2): 68-75.
- Meide van der PH et al. (1995). Enumeration of lymphokine-secreting cells as a quantitative measure for cellular immune responses in rhesus macaques. J Med Primatol 24: 271-281.
- Filbert H et al. (2013). Serum-free freezing media support high cell quality and excellent ELISPOT assay performance across a wide variety of different assay protocols. Cancer Immunol Immunother 62: 615–627.
- Janetzki S et al. (2008). Results and harmonization guidelines from two large-scale international ELISPOT proficiency panels conducted by the cancer vaccine consortium (cvc/svi). Cancer Immunol Immunother 57: 303–315.
- Santos R et al. (2015). Improvement of IFNγ ELISPOT performance following overnight resting of frozen PBMC samples confirmed through rigorous statistical analysis. Cells 4: 1-18.
- Donaldson MM et al. (2012). Optimization and qualification of an 8-color intracellular cytokine staining assay for quantifying T cell responses in rhesus macaques for pre-clinical vaccine studies. J Immunol Methods 14; 386: 10–21.
- Kuerten S et al. (2012). Resting of cryopreserved PBMC does not generally benefit the performance of Antigen-Specific T Cell ELISPOT Assays. Cells 1: 409-427.
- Gad E et al. (2013). Optimizing the cryopreservation of murine splenocytes for improved antigen- specific T cell function in ELISPOT. J Immunother Cancer 1: 211.