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Immunospot s4 pro analyzer

Manufactured by Cellular Technology
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The ImmunoSpot S4 Pro Analyzer is a compact, automated instrument designed for the detection and enumeration of antigen-specific T cells and B cells. The system utilizes the ELISPOT (Enzyme-Linked Immunospot) technique to quantify the number of cells secreting specific cytokines or antibodies in response to a given stimulus.

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Lab products found in correlation

Immunospot version 4, by Cellular Technology (2 mentions) Elispot kit, by R&D Systems (1 mentions) Elispot plate, by BD (1 mentions) Pan t cell isolation kit 2, by Miltenyi Biotec (1 mentions) Prism 6, by GraphPad (1 mentions) Human ifn γ elispot kit, by R&D Systems (1 mentions) Elispot assay, by R&D Systems (1 mentions) Immunospot software, by Cellular Technology (1 mentions)

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ImmunoSPOT S4 ImmunoSpot Analyzer

8 protocols using immunospot s4 pro analyzer

1

Quantifying Epitope-specific CD8+ T Cells

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Epitopes corresponding to reported antigenic peptides presented by MHC class-I molecules Kd, Dd, and Ld are derived from the mCMV open reading frames (ORFs) m04, m18, M45, M83, M84, M105, m123/IE1, m145, and m164 (listed in [31 (link),56 ]; for an update see [60 (link)]). Custom peptide synthesis with a purity of >80% was performed by JPT Peptide Technologies (Berlin, Germany).
At indicated times after intraplantar infection, CD8+ T cells were immunomagnetically purified from spleen cell suspensions (pool of 5–10 mice per time of analysis) to serve as responder cells in an IFNγ-based enzyme-linked immunospot (ELISpot) assay ([65 (link),69 (link)], and references therein). For quantitating functional, epitope-specific CD8+ T cells, synthetic peptides were exogenously loaded at the indicated molar concentrations on P815 (H-2d) mastocytoma cells for serving as stimulator cells in the assay. In brief, graded numbers of CD8+ T cells were seeded with the peptide-loaded stimulator cells in triplicate microcultures. After 18 hrs of incubation, spots, each representing a specifically sensitized IFNγ-secreting cell, were counted automatically, based on standardized criteria using ImmunoSpot S4 Pro Analyzer (Cellular Technology Limited, Cleveland, OH, USA).
Freitag K., Hamdan S., Reddehase M.J, & Holtappels R. (2021). Immunodominant Cytomegalovirus Epitopes Suppress Subdominant Epitopes in the Generation of High-Avidity CD8 T Cells. Pathogens, 10(8), 956.
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2

Analyzing B and T Cell Immunity to HspA and MV

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B and T cell-mediated immunity to HspA and MV antigens was analyzed on isolated spleen lymphocytes of immunized Ifnarko-CD46Ge mice using mouse IFN-γ and IgM/IgG dual-color B cell ELISPOT kits (R&D Systems). Spleen cells were harvested, washed, and plated (5 × 106 cells/well in 1 mL of culture medium) in 24-well plates for stimulation with MV-NIS at an MOI of 0.1, 100 ng/mL purified HspA, and corresponding empty pET-28a control extracts overnight. On the next day 5 × 104 and 5 × 105 cells in 100 μL of medium were transferred to the IFN-γ ELISPOT plates and incubated for 24 or 72 h before the analysis. For the HspA-specific IgM/IgG B cell response detection, spleen cells were transferred to HspA (200 ng/well) or control antigen-coated ELISPOT plates and cultured for 3 days. The reaction was developed following the ELISPOT kit instructions (R&D Systems). Results were analyzed on an ImmunoSpot S4 Pro analyzer (Cellular Technology, Cleveland, OH, USA) using ImmunoSpot version 4.0 software (Cellular Technology).
Iankov I.D., Kurokawa C., Viker K., Robinson S.I., Ammayappan A., Panagioti E., Federspiel M.J, & Galanis E. (2020). Live Attenuated Measles Virus Vaccine Expressing Helicobacterpylori Heat Shock Protein A. Molecular Therapy Oncolytics, 19, 136-148.
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3

T Cell Cytokine Production Analysis

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Interferon gamma and IL-17 ELISPOT (eBioscience) kits and ELISPOT plates from BD Biosciences were used for all ELISPOT assays. ELISPOT plates were prepared according to manufacturer's instructions. Primary MLR was performed and after 48 h responder T cells were purified by negative selection using the Pan T Cell Isolation Kit II (Miltenyi). These cells were rested over 24 h and subsequently, 5 × 104 CD4+ cells from each fraction were challenged with the same number of APC from the original stimulators, in 100 μl of complete medium. Production of IFN-γ and IL-17 was determined by ELISPOT after 48 h of culture. The numbers of spots were counted using ImmunoSpot S4 Pro Analyzer (Cellular Technology Ltd., Cleveland, OH) and the means of triplicate wells for each culture condition were calculated.
Nellore A., Liu B., Patsoukis N., Boussiotis V.A, & Li L. (2014). The cyclin dependent kinase inhibitor (R)-roscovitine mediates selective suppression of alloreactive human T cells but preserves pathogen-specific and leukemia-specific effectors. Clinical immunology (Orlando, Fla.), 152(0), 48-57.
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4

Quantifying Epitope-Specific CD8 T-cell Dynamics

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For longitudinal immune response analyses, groups of age-matched mice were randomized before treatment, and data for the indicated read-out times after infection represent the experimental average for pooled samples. Frequencies (most probable numbers) of cells responding in the ELISpot assay (see above) and the corresponding 95% confidence intervals were calculated by intercept-free linear regression analysis from the linear portions of regression lines, based on spot counts from triplicate assay cultures for each of the graded cell numbers seeded [38 (link)]. Spots were counted automatically based on standardized criteria using ImmunoSpot S4 Pro Analyzer (Cellular Technology Limited, Cleveland, OH, USA). For analyzing the dynamics of epitope-specific CD8 T-cell subpopulations, a trend analysis by linear regression was performed with Graph Pad Prism 6.04 (Graph Pad Software, San Diego, CA, USA). Rising and declining trends are reflected by positive and negative slopes of regression lines, respectively. Trends were considered as statistically significant by non-overlapping 95% confidence intervals and p-values of < 0.05, compared to the ‘null hypothesis’ of having no slope.
Holtappels R., Freitag K., Renzaho A., Becker S., Lemmermann N.A, & Reddehase M.J. (2020). Revisiting CD8 T-cell ‘Memory Inflation’: New Insights with Implications for Cytomegaloviruses as Vaccine Vectors. Vaccines, 8(3), 402.
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5

Quantifying Epitope-Specific CD8+ T Cells

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At the indicated time during viral latency established after HCT and infection, cells isolated from the different lung compartments served as responder cells in an IFNγ-based enzyme-linked immunospot (ELISpot) assay ([65 (link)] and references therein). Briefly, to detect functional, epitope-specific CD8+ T cells, synthetic peptides were exogenously loaded at a saturating concentration of 10−7M on P815 (H-2d) mastocytoma cells for serving as stimulator cells in the assay. Graded numbers of leucocytes were seeded with the peptide-loaded stimulator cells in triplicate microcultures. After 18 h of coculture, spots, each representing an IFNγ-secreting cell, were counted automatically, based on standardized criteria using ImmunoSpot S4 Pro Analyzer (Cellular Technology Limited, Cleveland, USA).
Blaum F., Lukas D., Reddehase M.J, & Lemmermann N.A. (2021). Localization of Viral Epitope-Specific CD8 T Cells during Cytomegalovirus Latency in the Lungs and Recruitment to Lung Parenchyma by Airway Challenge Infection. Life, 11(9), 918.
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6

Quantifying IFNγ-Producing Cells by ELISPOT

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Human IFNγ ELISPOT kits (R&D Systems; Minneapolis, MN) were used to measure the number of IFNγ-producing cells, as previously described [16 (link),44 (link),45 (link)], following the manufacturer’s protocol. We stimulated subjects’ PBMCs (or, alternatively, left them unstimulated) in triplicate with the Edmonston strain of MV (multiplicity of infection, MOI = 0.5), and developed the reaction after 42 h incubation at 37 °C, in 5% CO2. PHA (5 μg/mL) was used as a positive control. All plates were scanned and analyzed using the same counting parameters on an ImmunoSpot® S4 Pro Analyzer (Cellular Technology Ltd.; Cleveland, OH, USA) using ImmunoSpot® version 4.0 software (Cellular Technology Ltd.). The ELISPOT response is presented in spot-forming units (SFUs) per 2 × 105 cells (median MV-specific stimulated response from triplicate measurements, minus median unstimulated response from triplicate measurements).
Haralambieva I.H., Simon W.L., Kennedy R.B., Ovsyannikova I.G., Warner N.D., Grill D.E, & Poland G.A. (2015). Profiling of Measles-Specific Humoral Immunity in Individuals Following Two Doses of MMR Vaccine Using Proteome Microarrays. Viruses, 7(3), 1113-1133.
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7

ELISPOT Protocol for Measuring PBMC IFN-γ Secretion

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Enzyme-linked immunosorbent spot (ELISPOT) assays (R&D Systems) were performed to measure total peripheral blood mononuclear cell (PBMC) IFN-γ and CD8α+ IFN-γ secretion, as previously reported (Ovsyannikova et al. 2013a (link); Ovsyannikova et al. 2011 (link)). ELISPOT assays on the discovery cohort (total PBMC IFN-γ ELISPOT, n=1,058; CD8α+ IFN-γ ELISPOT, n=1,002) were performed six years prior to that of replication cohort (total PBMC IFN-γ ELISPOT, n=1,048; CD8α+ IFN-γ ELISPOT, n=991). PBMCs were stimulated for 24 hours with inactivated VACV at a multiplicity of infection (MOI) of 5 and an MOI of 1 for the discovery and replication cohorts, respectively. These parameters were set based upon the results of optimization performed prior to each independent study. Outcomes are expressed as spot-forming cells (SFC) per 2 × 105 PBMCs. Plates were scanned, and spots were counted with an ImmunoSpot S4 Pro-Analyzer using ImmunoSpot software, version 4.0 (Cellular Technology). Intraclass correlations ranged between 0.60 (total PBMC IFN-γ ELISPOT) to 0.63 (CD8α+ IFN-γ ELISPOT).
Ovsyannikova I.G., Pankratz V.S., Salk H.M., Kennedy R.B, & Poland G.A. (2014). HLA Alleles Associated with the Adaptive Immune Response to Smallpox Vaccine: A Replication Study. Human genetics, 133(9), 1083-1092.
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8

Epitope-specific CD8+ T Cell Quantification

Cited 1 time
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An 18-h IFNγ-based enzyme-linked immunospot (ELISpot) assay was used to determine the frequency of epitope-specific CD8+ T cells [(30 (link), 57 (link)) and references therein]. Briefly, graded numbers of immune-magnetically purified CD8+ T cells were sensitized in triplicate assay cultures by EL-4 (H-2b) cells that were exogenously loaded with epitope-representing synthetic peptides at the indicated molar loading concentrations. Custom peptide synthesis with a purity of >80% was performed by JPT Peptide Technologies (Berlin, Germany). Spots, representing single cells stimulated to secrete IFNγ, were counted automatically, based on standardized criteria using ImmunoSpot S4 Pro Analyzer (Cellular Technology Limited, Cleveland, OH, USA). Avidity distributions were derived by calculation from frequencies of CD8+ T cells responding to stimulation by graded target cell loading concentrations of synthetic peptides (cumulative avidity distribution), as explained in greater detail previously (55 (link), 58 (link)).
Gergely K.M., Podlech J., Becker S., Freitag K., Krauter S., Büscher N., Holtappels R., Plachter B., Reddehase M.J, & Lemmermann N.A. (2021). Therapeutic Vaccination of Hematopoietic Cell Transplantation Recipients Improves Protective CD8 T-Cell Immunotherapy of Cytomegalovirus Infection. Frontiers in Immunology, 12, 694588.
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