Immunospot elispot reader

Manufactured by Cellular Technology

Sourced in United States

The ImmunoSpot ELISPOT reader is a laboratory instrument designed for the detection and analysis of cytokine-secreting cells using the ELISPOT assay technique. It provides precise quantification of the number of cells secreting a specific cytokine in response to an immunological stimulus.

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Lab products found in correlation

Streptavidin horseradish peroxidase, by BD (2 mentions) Pvdf multiscreen hts ip plates, by Merck Group (2 mentions) Biotinylated mouse anti rabbit igg, by Southern Biotech (2 mentions) 3 3 diaminobenzidine substrate, by Merck Group (2 mentions) Sa alkaline phosphatase, by Southern Biotech (2 mentions) Vector blue substrate kit, by Vector Laboratories (2 mentions) Elispot plate, by Merck Group (2 mentions) Negative magnetic selection, by STEMCELL (1 mentions) Elispot assay, by R&D Systems (1 mentions) Mmessage t7 ultra transcription kit, by Thermo Fisher Scientific (1 mentions) Mouse granzyme b elispot kit, by R&D Systems (1 mentions) Concanavalin a (cona), by Merck Group (1 mentions) Hrp conjugated anti mouse igg iga antibody, by Southern Biotech (1 mentions) Multiscreen 96 well filtration plate, by Merck Group (1 mentions) Streptavidin sa hrp, by Southern Biotech (1 mentions) Pha m, by Thermo Fisher Scientific (1 mentions)

9 protocols using immunospot elispot reader

JHMV-specific IgG ASC Quantification

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JHMV-specific IgG ASC were measured by ELISPOT assay as previously described (DiSano et al., 2017 (link)). Briefly, 96-well PVDF MultiScreen HTS IP plates (EMD Millipore, Billerica, MA) were coated with JHMV DM (∼5 × 10⁵ PFU/well) overnight at 4 °C. Serial dilutions of cells plated in triplicate were incubated for 4 h at 37 °C and 5% CO₂. ASC was detected by sequential incubation with biotinylated rabbit anti-mouse IgG (0.5 μg/ml; Southern Biotech, Birmingham, AL) overnight at 4 °C, streptavidin horseradish peroxidase (1:1000; BD Biosciences, St. Louis, MO) for 1 h at room temperature, and filtered 3,3′-diaminobenzidine substrate (Sigma-Aldrich, St. Louis, MO) in 0.3% hydrogen peroxide. Brown spots were visible within 2–4 min and the reaction was terminated using cold tap water. Spots were counted using an ImmunoSpot ELISPOT reader (Cellular Technology Ltd., Shaker Heights, OH). Minimum and maximum spot size cutoffs were set to 0.0009 mm² and 0.2295 mm², respectively and spots were analyzed using diffuse processing and spot separation size of 3.00-5.00. Following automated counting, wells were re-counted manually for exclusion of artifacts. Wells containing ≥4 spots scored positive for virus-specific ASC. For analysis, 3–5 wells within a linear dilution range were averaged for each stimulation condition.

DiSano K.D., Stohlman S.A, & Bergmann C.C. (2017). An optimized method for enumerating CNS derived memory B cells during viral-induced inflammation. Journal of Neuroscience Methods, 285, 58-68.

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Mouse IgG ELISpot Assay

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ELISpot plates (Millipore) were coated with 100 HAU WIV overnight and blocked with cRPMI-1640 media. Dilutions of cells were added to plates and incubated overnight at 37°C. Plate-bound Abs were probed by biotin-α-mouse IgG, SA-alkaline phosphatase (Southern Biotech), then Vector Blue Substrate Kit (Vector Lab). Spots were counted using an ImmunoSpot^® ELISPOT reader (Cellular Technology Ltd.).

Kim J.H., Liepkalns J., Reber A.J., Lu X., Music N., Jacob J, & Sambhara S. (2015). Prior infection with influenza virus but not vaccination leaves a long-term immunological imprint that intensifies the protective efficacy of antigenically drifted vaccine strains. Vaccine, 34(4), 495-502.

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Expanded Papilloma T Cell Reactivity

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T lymphocyte cultures were initiated from fresh pretreatment and post-treatment papilloma fragments using interleukin-2 containing media. Autologous B lymphocytes were isolated from the Peripheral blood mononuclear cells (PBMC) using negative magnetic selection (StemCell) and expanded in the presence of irradiated (6000 rad) NIH3T3-CD40L feeder cells (1:1 ratio) and rhIL-4 (200 U/mL). Expanded papilloma T lymphocytes were assayed for reactivity against autologous B lymphocytes (1:2 ratio) electroporated with in vitro transcribed full-length mRNA from HPV 6 or 11 E2, E6 or E7 (mMESSAGE T7 ULTRA Transcription Kit, Thermo). T lymphocyte reactivity against these products or PMA/Ionomycin (positive control) was measured via ELISpot assay (R&D Systems). Spot counts were measured on an Immunospot ELISpot reader (Cellular Technology).

Robbins Y., Friedman J., Clavijo P.E., Sievers C., Bai K., Donahue R.N., Schlom J., Sinkoe A., Abdul Sater H., Gulley J.L., Norberg S., Hinrichs C.S, & Allen C. (2021). Dual PD-L1 and TGF-b blockade in patients with recurrent respiratory papillomatosis. Journal for Immunotherapy of Cancer, 9(8), e003113.

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B16F10 Tumor Cytotoxicity Assay

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B16F10 tumor-bearing mice were euthanized and spleens removed. Effector cells from splenocytes or the positive activation control ConA at 5 mg/mL (Sigma-Aldrich, St Louis, MO, USA) were incubated with B16F10 target cells for 48 h in 96-well plates in triplicate [12 (link)]. Granzyme B enzyme-linked immune spot (ELISPOT) assays were performed according to manufacturer’s protocol using a commercial Mouse Granzyme B ELISPOT kit (R&D system, Minneapolis, MN, USA). The plates were scanned and analyzed on an ImmunoSpot ELISPOT Reader (Cellular Technology Limited, Clevland, OH, USA).

Shi G., Edelblute C., Arpag S., Lundberg C, & Heller R. (2018). IL-12 Gene Electrotransfer Triggers a Change in Immune Response within Mouse Tumors. Cancers, 10(12), 498.

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JHMV-specific IgG Antibody-Secreting Cell Assay

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JHMV-specific IgG ASC were measured by ELISPOT assay as previously described (34 (link)). Briefly, 96-well PVDF MultiScreen HTS IP plates (EMD Millipore, Billerica, MA) were coated with JHMV DM (∼5 × 10⁵ PFU/well) overnight at 4°C. Serial dilutions of cells plated in triplicate were incubated for 4 hour at 37°C and 5% CO2. ASC was detected by sequential incubation with biotinylated rabbit anti-mouse IgG (0.5 μg/ml; Southern Biotech, Birmingham, AL) overnight at 4°C, streptavidin horseradish peroxidase (1:1000; BD Biosciences, St. Louis, MO) for 1 h at room temperature, and filtered 3,3′-diaminobenzidine substrate (Sigma-Aldrich, St. Louis, MO) in 0.3% hydrogen peroxide. Brown spots were visible within 2–4 minutes and the reaction was terminated using cold tap water. Spots were counted using an ImmunoSpot ELISPOT reader (Cellular Technology Ltd., Shaker Heights, OH). Minimum and maximum spot size cutoffs were set to .0009 mm² and .2295 mm², respectively and spots were analyzed using diffuse processing and spot separation size of 3.00–5.00. Following automated counting, wells were re-counted manually for exclusion of artifacts. Wells containing ≥4 spots scored positive for virus-specific ASC. For analysis, 3–5 wells within a linear dilution range were averaged for each stimulation condition.

DiSano K.D., Stohlman S.A, & Bergmann C.C. (2017). An optimized method for enumerating CNS derived memory B cells during viral-induced inflammation. Journal of Neuroscience Methods, 285, 58-68.

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Measuring Antigen-Specific Memory B Cells

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Antigen (Ag)–specific memory B cells were assessed in PBMCs collected 28 days after vaccination, and plasmablasts were derived from preexisting memory B cells assessed in samples obtained 7 days after vaccination. PBMCs stimulated to induce polyclonal activation (Supplementary Figure 1) were added to enzyme-linked immunospot (ELISPOT) plates coated with anti-human immunoglobulin G (IgG), immunoglobulin M (IgM; Southern Biotech, Birmingham, Alabama), or monovalent influenza vaccines (kindly provided by Sanofi Pasteur, Swiftwater, Pennsylvania). Spot-forming units were assessed by ImmunoSpot ELISPOT reader (Cellular Technology, Cleve-land, Ohio) and expressed as the percentage of Ag-specific IgG-or IgM-secreting B cells out of the total number of IgG- or IgM-secreting B cells.

Reber A.J., Kim J.H., Coleman L.A., Spencer S.M., Chung J.R., Chen J., Gargiullo P., Sundaram M.E., Belongia E.A., Shay D.K., Katz J.M, & Sambhara S. (2016). Seasonal Influenza Vaccination of Children Induces Humoral and Cell-Mediated Immunity Beyond the Current Season: Cross-reactivity With Past and Future Strains. The Journal of infectious diseases, 214(10), 1477-1486.

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Quantifying Virus-Specific Antibody-Secreting Cells

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For estimating virus-specific IgG and IgA ASCs, Multiscreen 96-well filtration plates (Millipore, Bedford, MA, USA) were coated with inactivated Aichi virus at 2 µg per ml in PBS and incubated overnight at 4 °C. Coated plates were washed with 0.05% Tween 20 in PBS (PBST) before 200 µl of RPMI 1640 with 10% FCS was added to each well for 2 h at 37 °C to block non-specific binding. Freshly prepared cells from spleen (SP), lung (LG), mediastinal lymph nodes (LN), and Peyer’s patches (PP) at a concentration of 1 × 10⁶ cells per ml, were suspended in 100 µl of complete RPMI medium and added to each well. After overnight incubation at 37 °C, plates were overlaid with 50 µl (2 µg per ml) of HRP-conjugated anti-mouse IgG/IgA antibody (Southern Biotech, Birmingham, AL, USA) and incubated for 1 h (1:1000 in PBST). After extensive washing, 3-3'-diaminobenzidine tetrahydrochloride (DAB; Research Genetics Inc., Huntsville, AL, USA) was added to develop spots in the plates (42 (link)). The plates were rinsed with water and air dried before counting using an ImmunoSpot ELISPOT reader (Cellular Technology, Shaker Heights, OH, USA).

Mohan T., Kim J., Berman Z., Wang S., Compans R.W, & Wang B.Z. (2016). Co-delivery of GPI-anchored CCL28 and influenza HA in chimeric virus-like particles induce cross-protective immunity against H3N2 viruses. Journal of controlled release : official journal of the Controlled Release Society, 233, 208-219.

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Influenza Antibody Binding and ASC Quantification

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Microneutralization (MN), HI assay were previously described78 (link). Sera and mucosal (BAL and nasal washes) Abs binding to influenza viruses were analyzed by ELISA. Briefly, Nunc 96 well plates (Maxi-sorb) were coated with 100 HAU WIV or 2 μg/ml His-tagged rHA proteins, then blocked with 4% BSA in PBS-Tween for 1 hr. Two or ten-fold dilutions of samples are added to the plates for 2 hr at room temperature or overnight at 4 °C. Plates were then developed by biotin-anti-mouse IgG/IgA followed by streptavidin (SA)-HRP (Southern Biotech). The signals were developed using 1 x TMB (ebioscience) and measured at 450 nm using a plate reader (Biotek). Ab binding strength was measured by biolayer interferometry on an Octet Red instrument (Fortebio, Inc.) using H1N1 recombinant HAs as previously described79 (link). The frequency of ASCs in spleen was measured by ELISpot assay as previously described80 (link). Briefly, ELISpot plates (Millipore) were coated with 100 HAU WIV overnight and blocked with cRPMI-1640 media. Dilutions of cells were added to plates and incubated overnight at 37 °C. Plate-bound Abs were probed by biotin-anti-mouse IgG, SA-alkaline phosphatase (Southern Biotech), then Vector Blue Substrate Kit (Vector Lab). Spots were counted using an ImmunoSpot^® ELISPOT reader (Cellular Technology Ltd.).

Kim J.H., Reber A.J., Kumar A., Ramos P., Sica G., Music N., Guo Z., Mishina M., Stevens J., York I.A., Jacob J, & Sambhara S. (2016). Non-neutralizing antibodies induced by seasonal influenza vaccine prevent, not exacerbate A(H1N1)pdm09 disease. Scientific Reports, 6, 37341.

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Interferon-Gamma ELISpot Assay

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ELISpots were performed using the Ready-Set-Go kit for IFN-𝛾 (eBioscience). Single-cell suspensions in RPMI were prepared from isolated spleens and 10⁶ cells per well were added to PVDF ELISpot plates that were precoated with antimouse IFN-𝛾 antibody. Cells were incubated with either 10 μg mL⁻¹ of the relevant peptide or an irrelevant peptide (SIINFEKL) for 24 h at 37 °C. Unstimulated cells in RPMI served as a negative control. Positive controls contained 2% PHA-M (Gibco). IFN-𝛾 spots were developed following the manufacturer’s protocol. Plates were scanned and quantified using an Immunospot ELISpot reader and analysis software (Cellular Technology).

Neek M., Tucker J.A., Butkovich N., Nelson E.L, & Wang S.W. (2020). An Antigen-Delivery Protein Nanoparticle Combined with Anti-PD-1 Checkpoint Inhibitor Has Curative Efficacy in an Aggressive Melanoma Model. Advanced therapeutics, 3(12), 2000122.

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