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Immunospot reader

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The ImmunoSpot reader is a piece of laboratory equipment used for analyzing and quantifying immune responses. It is designed to detect and measure the secretion of specific proteins, such as cytokines, by individual cells. The ImmunoSpot reader utilizes a specialized detection method to provide accurate and reliable data on the cellular immune response.

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Lab products found in correlation

Immunospot software, by Cellular Technology (4 mentions) Multiscreen ip plate, by Merck Group (4 mentions) Alkaline phosphatase conjugated streptavidin, by Jackson ImmunoResearch (3 mentions) Bcip nbt substrate kit, by Bio-Rad (3 mentions) Elispot plate, by Merck Group (2 mentions) Interleukin 2 (il 2), by BD (2 mentions) 3 amino 9 ethylcarbazole, by Merck Group (2 mentions) Peroxidase conjugated rabbit anti mouse immunoglobulin, by Merck Group (2 mentions) Streptavidin hrp conjugate, by Roche (2 mentions) Ifn γ elispot mouse kits, by BD (2 mentions) Aec substrate reagent kit, by BD (2 mentions) Streptavidin peroxidase, by Merck Group (2 mentions) Pvdf membrane plates, by Merck Group (2 mentions) Hrp conjugated anti mouse igg, by Jackson ImmunoResearch (1 mentions) Multiscreen ip filter plate, by Merck Group (1 mentions) Mouse ifn γ elispotplus kit, by Mabtech (1 mentions) Pma ionomycin, by Merck Group (1 mentions) Anti ifn γ mab, by BD (1 mentions) Mouse igg1 isotype control clone mopc 21, by BioXCell (1 mentions) Facsaria 3, by BD (1 mentions)

Close spelling variants of this product

CTL Immunospot Reader (20 citations) ImmunoSpot plate reader (12 citations) Immunospot ELISpot plate reader (11 citations) ImmunoSpot ELISPOT reader (9 citations) CTL ImmunoSpot S6 Ultra reader (9 citations) ImmunoSpot S6 Ultimate reader (8 citations) ImmunoSpot Reader 5.0 (3 citations) ImmunoSpot S6 UV Reader (3 citations) ImmunoSpot S6 Micro Analyzer Elispot Reader (3 citations) C.T.L ImmunoSpot plate reader and counting (2 citations)

27 protocols using immunospot reader

1

Measuring Antigen-Specific T Cell and Antibody Responses

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The ELISpot assay is a sensitive tool to measure antigen-specific T cell responses and antigen-specific antibody-secreting cells. For examining T cell responses, Mouse IFN-γ ELISpotPLUS kit (MabTech, Sweden) was used according to the manufacturer’s protocol. The plates were read by the ImmunoSpot reader (Cellular Technology, USA).
For the identification of antigen-specific antibody-secreting cells, M2e peptide, LAH peptide pool or NP protein at a final concentration of 5 μg/mL was pre-coated to MultiScreen-IP filter plates (Millipore, USA) overnight. 5 × 105 cells isolated from spleen, lung or lymph node were added in triplicates to plates and then incubated at 37°C in 5% CO2 for 48 h. After washing with PBS, the plates were added HRP-conjugated anti-mouse IgG (1:20000, Jackson ImmunoResearch) at room temperature for 1 h. Spots were exposed by TMB solution and were read by the ImmunoSpot reader (Cellular Technology).
Xiong F., Zhang C., Shang B., Zheng M., Wang Q., Ding Y., Luo J, & Li X. (2023). An mRNA-based broad-spectrum vaccine candidate confers cross-protection against heterosubtypic influenza A viruses. Emerging Microbes & Infections, 12(2), 2256422.
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2

Identification of EBOV T Cell Epitopes

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The peptide epitopes of EBOV GP and VP40 proteins that are specific for CD8+ T lymphocytes in BALB/c mouse (GP-T1: LYDRLASTV; GP-T2: GPCAGDFAF; VP40-T1: YFTFDLTALK; VP40-T2: TSPEKIQAIM) were selected according to previous reports (Warfield et al., 2005 (link); Wu et al., 2012 (link)), and synthesized (GeneScript, Nanjing, China). After the preparation of single cell suspensions from mouse splenocytes, ELISPOT assays were performed in pre-coated 96-well plates (Dakewe Biotech, China). The antibody-coated plates were blocked with complete RPMI 1640 medium for 2 h at room temperature. After blocking, 100 μl of splenocytes suspension (1 × 106 cells/ml) containing different peptides (10 μg/ml) were added to each well. A positive control PMA/ionomycin (Sigma) and a media negative control were included in all assays. The plates were incubated for 24 h in a humidified incubator at 37°C, 5% CO2. Plates were then washed and processed according to manufacturer’s instructions, and spots were enumerated using an ImmunoSpot reader and ImmunoSpot software (Cellular Technology Ltd.). Peptide-specific CD8+ T-cell frequency was expressed as Spots forming cells (SFCs)/1 × 105 splenocytes. Background spots (negative control wells) were subtracted from test wells. A positive response to a peptide was defined as having > 5 SFCs/1 × 105 splenocytes after subtraction of the background.
Ren S., Wei Q., Cai L., Yang X., Xing C., Tan F., Leavenworth J.W., Liang S, & Liu W. (2018). Alphavirus Replicon DNA Vectors Expressing Ebola GP and VP40 Antigens Induce Humoral and Cellular Immune Responses in Mice. Frontiers in Microbiology, 8, 2662.
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3

Quantifying CD8+ T Cell and Anti-IDUA IgG Response

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CD8+ T cells were magnetically isolated from the spleen of experimental mice (Miltenyi Biotec, 130-104-075). 105 CD8+ T cells were plated in triplicate in ELISPOT plates (Millipore, Bedford, MA) pre-coated with anti-IFN-γ capture mAb (2.5 μg/mL; BD Pharmingen, R46A2) in the presence of IL-2 (50 U/mL; BD Pharmingen) and 105 irradiated (6,000 rad) untransduced or LV.IDUA-transduced autologous EL-4 cells. After 42 h of incubation at 37°C and 5% CO2, plates were washed and IFN-γ-producing cells were detected by biotin-conjugated anti-IFN-γ mAb (0.5 μg/mL; BD Pharmingen, XMG 1.2). Streptavidin-HRP conjugate (Roche) was added. Total splenocytes or total BM (0.35 × 106 cells/well) was plated in complete RPMI in triplicate in ELISPOT plates pre-coated with rhIDUA (2 μg/well). After 24 h of incubation at 37°C and 5% CO2, plates were washed and anti-IDUA IgG-secreting cells were detected with peroxidase-conjugated rabbit anti-mouse immunoglobulin (Sigma A2554). All plates were reacted with H2O2 and 3-Amino-9-ethylcarbazole (Sigma, A6926). Spots were counted by ImmunoSpot reader (Cellular Technology).
Squeri G., Passerini L., Ferro F., Laudisa C., Tomasoni D., Deodato F., Donati M.A., Gasperini S., Aiuti A., Bernardo M.E., Gentner B., Naldini L., Annoni A., Biffi A, & Gregori S. (2019). Targeting a Pre-existing Anti-transgene T Cell Response for Effective Gene Therapy of MPS-I in the Mouse Model of the Disease. Molecular Therapy, 27(7), 1215-1227.
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4

IFN-γ ELISPOT Assay for Measuring Immune Cell Responses

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Ifn-γ ELISPOT mouse kits (BD Biosciences) were used according to the manufacturer’s instructions. Briefly, 96-well filtration plates were coated overnight at 4°C with Ifn-γ capturing monoclonal antibody; the plates were then washed and blocked with RPMI-1640 medium containing 10% FBS for 2h at room temperature. Splenocytes and tumor-infiltrating immune cells (TIICs) isolated from different treatment groups were counted and resuspended in 10% FBS supplemented RPMI-1640. Splenocytes and TIICs were seeded into the 96-well filtration plate at concentrations of 1 × 106 cells/well and 2.5 × 104 cells/well, respectively. After mitomycin treatment, 5 × 104 C57BL/6J fat pad derived primary cells or E0771 tumor cells were added as different stimulators to stimulate the Ifn-γ secretion. The plates were cultured for approximately 45 hours at 37°C and 5% CO2. The co-culture were stopped by soaking in DI water, and washing three times with PBST; the plates were incubated with biotinylated detection antibody at room temperature for 2 h. Then, plates were incubated with HRP-conjugated streptavidin at room temperature for 1 h after washing three times with PBST. Spots were revealed using an AEC substrate reagent kit (BD Bioscience) at room temperature and counted using an Immunospot Reader (Cellular Technology). Statistical significance was assessed by two-tailed unpaired Welch’s t-test.
Wang G., Chow R.D., Bai Z., Zhu L., Errami Y., Dai X., Dong M.B., Ye L., Zhang X., Renauer P.A., Park J.J., Shen L., Ye H., Fuchs C.S, & Chen S. (2019). Multiplexed activation of endogenous genes by CRISPRa elicits potent anti-tumor immunity. Nature immunology, 20(11), 1494-1505.
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5

IFN-γ ELISPOT Assay for Measuring Immune Cell Responses

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Ifn-γ ELISPOT mouse kits (BD Biosciences) were used according to the manufacturer’s instructions. Briefly, 96-well filtration plates were coated overnight at 4°C with Ifn-γ capturing monoclonal antibody; the plates were then washed and blocked with RPMI-1640 medium containing 10% FBS for 2h at room temperature. Splenocytes and tumor-infiltrating immune cells (TIICs) isolated from different treatment groups were counted and resuspended in 10% FBS supplemented RPMI-1640. Splenocytes and TIICs were seeded into the 96-well filtration plate at concentrations of 1 × 106 cells/well and 2.5 × 104 cells/well, respectively. After mitomycin treatment, 5 × 104 C57BL/6J fat pad derived primary cells or E0771 tumor cells were added as different stimulators to stimulate the Ifn-γ secretion. The plates were cultured for approximately 45 hours at 37°C and 5% CO2. The co-culture were stopped by soaking in DI water, and washing three times with PBST; the plates were incubated with biotinylated detection antibody at room temperature for 2 h. Then, plates were incubated with HRP-conjugated streptavidin at room temperature for 1 h after washing three times with PBST. Spots were revealed using an AEC substrate reagent kit (BD Bioscience) at room temperature and counted using an Immunospot Reader (Cellular Technology). Statistical significance was assessed by two-tailed unpaired Welch’s t-test.
Wang G., Chow R.D., Bai Z., Zhu L., Errami Y., Dai X., Dong M.B., Ye L., Zhang X., Renauer P.A., Park J.J., Shen L., Ye H., Fuchs C.S, & Chen S. (2019). Multiplexed activation of endogenous genes by CRISPRa elicits potent anti-tumor immunity. Nature immunology, 20(11), 1494-1505.
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6

Quantifying IFN-γ-Producing Cells in PBMCs

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PBMC, BALC, and lymphoid tissue cells were plated at 2.5 × 105 cells/well in 96-well PVDF membrane plates (Merck Life Science) coated with anti-porcine IFN-γ mAb (clone P2G10; BD Pharmingen, Oxford, UK). Cells were either left unstimulated (cRPMI), stimulated with M-NSP5 peptide pool at 1 μg/mL, or stimulated with 5 µg/mL concanavalin A as a positive control. All experiments were performed in triplicate. The plates were incubated overnight at 37°C and 5% CO2. The cells were removed, and secondary biotinylated anti-porcine IFN-γ mAb (clone P2C11, BD Pharmingen) was added, followed by further washing. Plates were then developed with streptavidin–alkaline phosphatase and a BCIP/NBT colorimetric substrate (both Mabtech, 2BScientific, Kirtlington, UK). The spots were counted using an ImmunoSpot Reader (Cellular Technology Limited, Ohio, USA), and the results were expressed as the number of IFN-γ-producing cells per 106 cells minus the average number of IFN-γ-producing cells in unstimulated wells.
de Brito R.C., Holtham K., Roser J., Saunders J.E., Wezel Y., Henderson S., Mauch T., Sanz-Bernardo B., Frossard J.P., Bernard M., Lean F.Z., Nunez A., Gubbins S., Suárez N.M., Davison A.J., Francis M.J., Huether M., Benchaoui H., Salt J., Fowler V.L., Jarvis M.A, & Graham S.P. (2023). An attenuated herpesvirus vectored vaccine candidate induces T-cell responses against highly conserved porcine reproductive and respiratory syndrome virus M and NSP5 proteins that are unable to control infection. Frontiers in Immunology, 14, 1201973.
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7

Quantifying HBV-specific T Cell Responses

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PBMC samples from the 65 patients with chronic HBV were thawed, washed in CTL Anti-AggregateTM medium (Cellular Technology Limited, CTL) and left overnight at 37°C in complete RPMI with 10% human serum. Cells were plated at 1E7/ml and overlapping peptide pool for the HBV-capsid protein (≫90% purity, JPT) was added to the culture. IL-2 at a final concentration of 2 IU/ml was added to the cells at day 2 and maintained until day 5. After a 5-day stimulation, quadruplicates of 2.5E5 expanded cells were re-stimulated with the same HBV-capsid peptide pool (or actin peptide pool as irrelevant peptide, when appropriate) in the presence of MEDI2790 or a control IgG isotype. Staphylococcal enterotoxin B was included as a positive control in every plate. HBV-specific T cell responses were quantified by ELISpot using ELISpotPLUS interferon (IFN)-γ pre-coated plates (MabTech) according to manufacturer’s instructions. Quantification was performed using the ImmunoSpot® reader and images were analyzed with the ImmunoSpot® software (Cellular Technology Limited, CTL).
Ferrando-Martinez S., Huang K., Bennett A.S., Sterba P., Yu L., Suzich J.A., Janssen H.L, & Robbins S.H. (2019). HBeAg seroconversion is associated with a more effective PD-L1 blockade during chronic hepatitis B infection. JHEP Reports, 1(3), 170-178.
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8

IFN-γ ELISPOT Assay for Mycobacterial Antigens

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IFN-γ Elispot assay was performed as previously described [24 (link)], with some modifications. Briefly, multiscreen-IP plates (Millipore, Bedford, MA) were coated with anti-IFN-γ mAb (An-18, eBioscience) at 5μg/ml in PBS. BMDCs were pre-pulsed with Mtb fractions or recombinant protein antigens overnight. In a blocking assay, Mtb antigen-pulsed BMDCs were pre-incubated with mouse IgG or anti-Qa-2 mAb (20-8-4) [48 (link)] for 30 min before the assay. Enriched CD8+ T cells from infected mice (5×103−2×104) were mixed with BMDCs stimulator cells (5×104/well) in RPMI 10 medium and plated in triplicate wells. After 18h incubation at 37°C, plates were washed using PBS-Tween (PBS and 0.05% Tween 20) and incubated for 2h at room temperature with biotinylated anti-IFN-γ mAb (R4.6A2, eBioscience). Plates were then washed and incubated with streptavidin-conjugated alkaline phosphatase (Jackson ImmunoResearch Laboratories, West Grove, PA). After 1 h incubation at room temperature, plates were developed with a BCIP/NBT substrate kit (Bio-Rad, Hercules, CA) according to the manufacturer’s instructions. Spots were counted using an ImmunoSpot reader (Cellular Technology, Shaker Heights, OH).
Shang S., Siddiqui S., Bian Y., Zhao J, & Wang C.R. (2016). Nonclassical MHC Ib-restricted CD8+ T Cells Recognize Mycobacterium tuberculosis-Derived Protein Antigens and Contribute to Protection Against Infection. PLoS Pathogens, 12(6), e1005688.
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9

IFN-γ ELISpot Assay for CD8+ T Cell Response

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IFN-γ ELISpot assay was performed as previously described [11 (link)], with some modifications. Briefly, multiscreen-IP plates (Millipore, Bedford, MA) were coated with anti-IFN-γ mAb (AN-18, BioLegend, San Diego, CA) at 5μg/ml in PBS. Enriched CD8+ T cells from infected mice were incubated with MHC II-/- BMDCs with media alone or 5μM of Mtb peptide, in duplicate. To confirm Qa-1 restriction, BMDCs were pre-incubated with 2 μg/ml anti-Qa-1 blocking mAb (6A8.6F10.1A6, BD, Franklin Lakes, NJ) or mouse IgG1 isotype control (clone MOPC-21) (BioXCell, West Lebanon, NH) prior to adding peptide and lymphocytes. After 18h incubation at 37°C, plates were washed using PBS/0.05% Tween 20 and developed using biotinylated α-IFN-γ mAb (R4.6A2, eBioscience, San Diego, CA), followed by streptavidin-conjugated alkaline phosphatase (Jackson ImmunoResearch Laboratories, West Grove, PA) and a BCIP/NBT substrate kit (Bio-Rad, Hercules, CA) according to the manufacturer’s instructions. Spots were counted using an ImmunoSpot reader (Cellular Technology, Shaker Heights, OH).
Bian Y., Shang S., Siddiqui S., Zhao J., Joosten S.A., Ottenhoff T.H., Cantor H, & Wang C.R. (2017). MHC Ib molecule Qa-1 presents Mycobacterium tuberculosis peptide antigens to CD8+ T cells and contributes to protection against infection. PLoS Pathogens, 13(5), e1006384.
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10

Quantification of IFN-γ and Anti-IDUA Responses

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CD8+ T cells were magnetically isolated from the spleen (Miltenyi Biotec, 130-104-075). 105 CD8+ T cells were plated in triplicate in ELISPOT plates (Millipore, Bedford, MA) pre-coated with anti–IFN-γ capture monoclonal Ab (2.5 μg/mL; BD Pharmingen, R46A2) in the presence of IL-2 (50 U/mL; BD Pharmingen) and 105 irradiated (6000 rad) un-transduced or LV.Mecp2-transduced autologous EL-4 cells. After 42 hr of incubation at 37°C 5% CO2, plates were washed and IFN-γ–producing cells were detected by biotin-conjugated anti–IFN-γ monoclonal Ab (0.5 μg/mL; BD Pharmingen, XMG 1.2). Streptavidin-HRP conjugate (Roche) was added. Total splenocytes or total BM (0,35 × 106 cells/well) were plated in complete RPMI in triplicate in ELISPOT plates pre-coated with rhIDUA (2 µg/well). After 24 hr of incubation at 37°C 5% CO2, plates were washed and anti-IDUA IgG secreting cells were detected with peroxidase-conjugated rabbit anti–mouse immunoglobulin (Sigma-Aldrich, RRID:AB_258008). All plates were reacted with H2O2 and 3-Amino-9-ethylcarbazole (Sigma-Aldrich, RRID:AB_2767485). Spots were counted by ImmunoSpot reader (Cellular Technology Limited).
Luoni M., Giannelli S., Indrigo M.T., Niro A., Massimino L., Iannielli A., Passeri L., Russo F., Morabito G., Calamita P., Gregori S., Deverman B, & Broccoli V. (2020). Whole brain delivery of an instability-prone Mecp2 transgene improves behavioral and molecular pathological defects in mouse models of Rett syndrome. eLife, 9, e52629.
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