Automated elispot counter

Manufactured by Cellular Technology

Sourced in United States

The Automated ELISPOT Counter is a laboratory instrument designed to detect and quantify the number of cytokine-producing cells in a given sample. It employs the ELISPOT (Enzyme-Linked Immunospot) technique, which is a sensitive method for the detection of individual cells secreting specific proteins. The core function of the Automated ELISPOT Counter is to accurately and efficiently count the number of spots, representing the cytokine-secreting cells, in an ELISPOT assay.

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Lab products found in correlation

Streptomycin, by Thermo Fisher Scientific (1 mentions) 3 amino 9 ethylcarbazole, by Merck Group (1 mentions) Penicillin g, by Thermo Fisher Scientific (1 mentions) Elispot filter plates, by Merck Group (1 mentions) Goat anti human ig, by Jackson ImmunoResearch (1 mentions) Avidin d hrp conjugate, by Vector Laboratories (1 mentions) Streptavidin alkaline phosphatase, by Southern Biotech (1 mentions) Biotinylated anti human igg, by Mabtech (1 mentions) Nbt bcip, by Thermo Fisher Scientific (1 mentions) Avidin d hrp, by Vector Laboratories (1 mentions) Aec substrate, by Merck Group (1 mentions) Affinipure donkey anti human igg h l, by Jackson ImmunoResearch (1 mentions) Staphylococcus aureus cowan, by Merck Group (1 mentions) Polyvalent goat anti human ig, by Jackson ImmunoResearch (1 mentions) Avidin d horseradish peroxidase conjugate, by Vector Laboratories (1 mentions) 3 amino 9 ethylcarbazole substrate, by Merck Group (1 mentions)

5 protocols using automated elispot counter

Enumeration of Antigen-Specific Antibody-Secreting Cells

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A direct enzyme-linked immunospot (ELISpot) assay was used to enumerate the numbers of total IgG, IgA and IgM ASCs and the H3N2-specific IgG, IgA, and IgM ASCs present in fresh PBMCs, performed as described previously [10 (link)]. 96-well ELISpot filter plates (Millipore, Burlington, MA, USA), #MAHA N4510) were coated overnight with purified A/Minnesota/11/2010 H3N2v virus or rHAs of contemporary H3N2 strains in PBS, or goat anti-human Ig (Jackson ImmunoResearch Laboratory, Inc., West Grove, PA, USA). Plates were washed and blocked 2 h at 37 °C prior to use with RPMI 1640 containing 10% fetal calf serum, 100 units/mL of penicillin G, and 100 μg/mL of streptomycin (Gibco), referred to as complete medium. Freshly isolated PBMC were suspended in complete medium distributed into ELISpot plates and incubated overnight at 37 °C in a CO₂ incubator. ASCs were detected with biotinylated anti-human IgG, IgM, or IgA antibody (Jackson ImmunoResearch Laboratory Inc., West Grove, PA, USA) followed by incubation with Avidin-D-HRP conjugate (Vector Laboratories, Burlingame, CA, USA), and then developed using AEC substrate (3 amino-9 ethyl-carbazole; Sigma-Aldrich, St. Louis, MO, USA). Developed plates were scanned and analyzed using an automated ELISpot counter (Cellular Technologies, Ltd., Shaker Heights, OH, USA).

Lai L., Rouphael N., Xu Y., Sherman A.C., Edupuganti S., Anderson E.J., Lankford-Turner P., Wang D., Keitel W., McNeal M.M., Cross K., Hill H., Bellamy A.R, & Mulligan M.J. (2020). Baseline Levels of Influenza-Specific B Cells and T Cell Responses Modulate Human Immune Responses to Swine Variant Influenza A/H3N2 Vaccine. Vaccines, 8(1), 126.

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ELISPOT Assay for Antibody-Secreting Cells

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Filter plates (96-well; Millipore) were incubated overnight at 4°C with anti-human IgG, IgA, and IgM (KPL), 2μg/ml recombinant HAs, 2μg/ml recombinant NAs or 32 HAU/well of whole virus. The plates were washed and blocked by incubation with RPMI containing 10% FBS at 37°C for 2 hours. Freshly isolated human PBMCs or mouse spleen cells were washed three times and were re-suspended in medium (RPMI medium supplemented with 4 mM L-glutamine, 10 mM HEPES, 100 U/ml Pen/Step, 10% FBS). The cells (0.5–1-10^E6) underwent 2-fold serial dilutions before being transferred to the ELISPOT plates, which were incubated overnight at 37°C. The plates were washed extensively with PBS and PBS–0.05% Tween before antibody from the antibody-secreting cells (ASC) were detected with either 1:1000 biotinylated anti-human IgG (Mabtech) and biotinylated anti-human IgA (Southern Biotech) or a 1:10000 biotinylated anti-mouse IgG. After a 2 hour incubation at room temperature (RT), a 1:500 dilution of streptavidin-alkaline phosphatase (Southern Biotech) was added to each well. The incubation was repeated before ASC spots were revealed with nitroblue tetrazolium/5-bromo-4-chloro-3-indolylphosphate (NBT/BCIP; Thermo Scientific). The developed plates were scanned using an automated ELISPOT counter (Cellular Technologies, Ltd.).

Chen Y., Wohlbold T.J., Zheng N.Y., Huang M., Huang Y., Neu K.E., Lee J., Wan H., Rojas K.T., Kirkpatrick E., Henry C., Palm A.K., Stamper C.T., Lan L.Y., Topham D.J., Treanor J., Wrammert J., Ahmed R., Eichelberger M.C., Georgiou G., Krammer F, & Wilson P.C. (2018). Influenza infection in humans induces broadly cross-reactive and protective neuraminidase-reactive antibodies. Cell, 173(2), 417-429.e10.

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ELISPOT Assay for Human IgG Detection

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Ninety-six well filter plates (Millipore–MSHAN4B50) were coated with either VZV cell lysate (Meridian Life Sciences) at 5 μg/well or Affinipure donkey anti-human IgG (H+L; Jackson Immunoresearch) at 100 ng/well in PBS overnight at 4°C. The plates were washed with PBS four times and complete FBS media added for 2 hr at 37°C. Freshly prepared PBMCs were resuspended in complete FBS media at 1×10⁷/ml and added to each well, followed by threefold dilution and then incubated overnight at 37°C. The next day, the plates were washed four times with PBS, four times with PBS-T and incubated for 1.5 hr at room temperature with Biotin-SP-Affinipure F(ab′)₂ donkey anti-human IgG (Jackson Immunoresearch) at 100 ng/well in PBS + 10% FCS + 0.05% Tween 20 (PBS-T-FBS). Plates were washed four times with PBS-T and incubated for 1.5 hr with HRP-avidin D (Vector laboratories) at 1:1000 in PBS-T-FBS. Plates were washed four times with PBS-T, four times with PBS and then developed using AEC substrate (3 amino-9 ethyl-carbazole; Sigma Aldrich). Developed plates were dried, scanned and analyzed using an automated ELISPOT counter (Cellular Technologies, Ltd).

Li S., Sullivan N.L., Rouphael N., Yu T., Banton S., Maddur M.S., McCausland M., Chiu C., Canniff J., Dubey S., Liu K., Tran V., Hagan T., Duraisingham S., Wieland A., Mehta A., Whitaker J.A., Subramaniam S., Jones D.P., Sette A., Vora K., Weinberg A., Mulligan M.J., Nakaya H.I., Levin M., Ahmed R, & Pulendran B. (2017). Metabolic Phenotypes Of Response to Vaccination in Humans. Cell, 169(5), 862-877.e17.

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Quantifying Influenza-Specific Memory B Cells

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H3N2 A/Minnesota/11/2010 vaccine antigen was provided by Sanofi Pasteur. Recombinant HA (rHA) from H3N2 A/Perth/16/2009 (NR-19442) was provided by Biodefense and Emerging Infections Research Repository (available at: http://www.beiresources.org); rHA from H3N2 A/Victoria/361/2011 (IRR number, FR-1059) was provided by the IRR. At Emory University School of Medicine, cryopreserved peripheral blood mononuclear cells (PBMCs) collected on day 0 from a subset of participants were thawed in a 37°C water bath and washed. Cells were counted and checked for viability by Trypan blue dye exclusion. MBC assays were performed as previously described [16 (link), 17 (link)]. In brief, PBMCs were plated in 24-well dishes at 5 × 10⁵ cells/well in R-10 medium supplemented with an optimized mix of polyclonal mitogens: pokeweed mitogen extract (made in-house), phosphorothiolated CpG ODN-2006 (Integrated DNA Technologies), and Staphylococcus aureus Cowan (Sigma). Eight wells were cultured per individual for 6 days. Stimulated cells were harvested, washed, and assayed using the ELISpot assay. Developed plates were scanned and analyzed using an automated ELISpot counter (Cellular Technologies). Data are presented as the percentage of influenza virus–specific immunoglobulin G (IgG)–secreting cells among total IgG-secreting cells.

Keitel W.A., Jackson L.A., Edupuganti S., Winokur P.L., Mulligan M.J., Thornburg N.J., Patel S.M., Rouphael N.G., Lai L., Bangaru S., McNeal M.M., Bellamy A.R., Hill H.R., Bond N., Bosworth K., Brown J., Banay J., Cheesman C., Lanford T., Munoz F., Wells J., Carste B., Dunstan M., Mathis A., Parikh M., Phillips C.H., Spingola A., Starkovich P., Suyehira J., Beck A., Mask K., Bower M., Osinski E., Rimann N., Turner P., Wang D., Stapleton J., Won R., Wagner N., Dull G., Gerot N., Reidy M., Zhao D., Segar E., Slaughter J.C., McDonough M., He F., Salata R., Meissner C., Sheffield J., Spigarelli M., Greenberg S., Agrawal A., Dublin S., Arterburn D., Rimland D., Ribner B., Meier J., Buchanan W., Chang S., Lambert L., Murray S., Riddle V, & Spiro D. (2015). Safety and Immunogenicity of a Subvirion Monovalent Unadjuvanted Inactivated Influenza A(H3N2) Variant Vaccine in Healthy Persons ≥18 Years Old. The Journal of Infectious Diseases, 212(4), 552-561.

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Enumerating Influenza-Specific Plasmablasts

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ELISPOT was performed to enumerate influenza-specific plasmablasts present in PBMC samples. We coated 96-well ELISPOT assay mixed cellulose ester filter plates (Millipore) overnight with either the 2016/2017 Fluarix quadrivalent influenza (GlaxoSmithKline) at 1:20 in PBS or polyvalent goat anti-human Ig (Jackson ImmunoResearch, West Grove, PA, USA) at 10 μg/mL in PBS. The plates were washed and blocked by incubation with R10 media (RPMI-1640 supplemented with 10% FBS, penicillin, streptomycin, and l-glutamine) at 37 °C for 2 h. Freshly isolated PBMCs were added to the plates in a dilution series starting at 5 × 10⁵ cells and incubated overnight at 37 °C in R10 media. The plates were washed with PBS, followed by PBS/0.05% Tween, and then incubated with biotinylated anti-human IgG, IgA, or IgM antibody (Invitrogen) at room temperature for 90 min. After washing, the plates were incubated with avidin D-horseradish peroxidase conjugate (Vector Laboratories) and developed using 3-amino-9-ethylcarbazole substrate (Sigma-Aldrich). Plates were scanned and analyzed using an automated ELISPOT counter (Cellular Technology Limited (CTL)).

Upadhyay A.A., Kauffman R.C., Wolabaugh A.N., Cho A., Patel N.B., Reiss S.M., Havenar-Daughton C., Dawoud R.A., Tharp G.K., Sanz I., Pulendran B., Crotty S., Lee F.E., Wrammert J, & Bosinger S.E. (2018). BALDR: a computational pipeline for paired heavy and light chain immunoglobulin reconstruction in single-cell RNA-seq data. Genome Medicine, 10, 20.

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