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Ctl immunospot reader

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The CTL Immunospot Reader is a specialized laboratory instrument designed to analyze and quantify cellular immune responses. It measures the production of cytokines or other secreted molecules by individual cells, providing a precise assessment of the cellular immune function.

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Lab products found in correlation

Bcip nbt substrate, by Merck Group (3 mentions) Extravidin alkaline phosphatase, by Merck Group (2 mentions) Mouse ifn gamma elispot plus kits, by Mabtech (2 mentions) Elispot assay, by Mabtech (1 mentions) Il 10, by Immunotools (1 mentions) Interleukin 6 (il 6), by Immunotools (1 mentions) Interleukin 2 (il 2), by Immunotools (1 mentions) Immobilon p plates, by Merck Group (1 mentions) Np4 bsa, by Biosearch Technologies (1 mentions) Np33 bsa, by Biosearch Technologies (1 mentions) Immunosorb plates, by Thermo Fisher Scientific (1 mentions) Emax microplate reader, by Molecular Devices (1 mentions) Aec substrate solution, by Merck Group (1 mentions) Red blood cell lysis buffer, by Merck Group (1 mentions) 40 m cell strainer, by BD (1 mentions) Concanavalin a (cona), by Thermo Fisher Scientific (1 mentions) Akp streptavidin, by BD (1 mentions) Multiscreenhts plates, by Merck Group (1 mentions) Penicillin, by Merck Group (1 mentions) Streptomycin, by Merck Group (1 mentions)

Close spelling variants of this product

ImmunoSpot reader (27 citations) CTL ImmunoSpot S6 Ultra reader (9 citations)

20 protocols using ctl immunospot reader

1

Induction of IgM-Secreting Cells

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To induce immunoglobulin production IL-2, IL-6, and IL-10 (50 ng/ml, each, Immunotools, Friesoythe, Germany) were added to the cells in the presence of different combination of the activators and the CR1 ligand for 3 days at 37°C and 5% CO2, as shown in the figures. The frequency of IgM secreting cells was evaluated by enzyme-linked immunosorbent spot (ELISpot) assay according to the manufacturer's guide (MabTech, Nacka Strand, Sweden). Briefly, 96-well Multiscreen-IP Filter Plates (MAIPSWU10, Millipore, Burlington, MA, USA) were coated with 15 μg/ml mouse anti-human IgM (MabTech, Nacka Strand, Sweden) at 4°C, overnight. After washing, 1 × 104 differently treated cells were added to the wells, and incubated at 37°C. After 20 h cells were aspirated and plates were washed and incubated with 1 μg/ml biotinilated mouse anti-human IgM (Mabtech, Nacka Strand, Sweden) detection antibody, followed by horse raddish peroxidase conjugated streptavidin (MabTech, Nacka Strand, Sweden). Spots were developed with 3,3′,5,5′-tetramethylbenzidine (TMB, MabTech, Nacka Strand, Sweden), and counted using the CTL Immunospot Reader (Cellular Technologies, Shaker Heights, OH, USA).
Mácsik-Valent B., Nagy K., Fazekas L, & Erdei A. (2019). Complement Receptor Type 1 (CR1, CD35), the Inhibitor of BCR-Mediated Human B Cell Activation, Differentially Regulates TLR7, and TLR9 Induced Responses. Frontiers in Immunology, 10, 1493.
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2

NP-Specific Antibody Detection ELISA/ELISPOT

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Immunosorb plates (Nunc) or Immobilon-P plates (Millipore) were coated with 10 µg/ml NP4-BSA or NP33-BSA (Biosearch Technologies) for ELISA or ELISPOT, respectively, and blocked with PBS containing 2% BSA. For ELISA, sera were incubated for 2 h at room temperature and plates developed with horseradish peroxidase–conjugated anti–mouse IgM or IgG1 antibodies (SouthernBiotech) using 3,3′, 5,5′ tetramethylbenzidine (TMB) substrate (BD). Color development was terminated with 2N H2SO4 then read on an EMax microplate reader (Molecular Devices). Purified anti-NP IgM or anti-NP IgG1 used as standards were a gift from G. Kelsoe (Duke University). For ELISPOTs, splenocyte suspensions were incubated for 4–5 h in RPMI/DMEM containing 10% FBS. Plates were developed with biotin-conjugated anti–mouse IgM or IgG1 antibodies (SouthernBiotech), followed by ExtrAvidin-Alkaline Phosphatase (Sigma-Aldrich) using NBT/BCIP substrate (Sigma-Aldrich), and color development was terminated with 1 M NaH2PO4. Spots were enumerated on CTL-ImmunoSpot reader (Cellular Technologies).
Goenka R., Matthews A.H., Zhang B., O’Neill P.J., Scholz J.L., Migone T.S., Leonard W.J., Stohl W., Hershberg U, & Cancro M.P. (2014). Local BLyS production by T follicular cells mediates retention of high affinity B cells during affinity maturation. The Journal of Experimental Medicine, 211(1), 45-56.
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3

SARS-CoV-2 Spike Protein T-cell Response Assay

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Cat IFN-γ ELISpotPLUS kits (MabTech) were used to determine SARS-CoV-2 S antigen-specific T lymphocyte response by enzyme-linked immunospot (ELISpot). The epitopes within S protein were predicted (http://www.iedb.org/), and 79 peptides (14 amino acids per peptide except for a peptide carrying 18 amino acids) were synthesized by Guangzhou IGE Biotechnology (Table S6). Each peptide was dissolved in water or DMSO to obtain a 5 mg/mL stock solution, and all peptides were mixed equally, aliquoted, and stored at −80°C. Peptide aliquots were thawed once and used immediately. PBMCs (1 × 105 cells/well) were stimulated with S peptide mixture (5 μg/mL) in duplicate and incubated with 5% CO2 for 48 h at 37°C. Spots were developed according to the kit’s instruction and then counted with a CTL Immunospot Reader (Cellular Technology). The results were expressed as SFCs per million cells.17 (link),25 (link)
Zhang P., Luo S., Zou P., Liang C., Wang C., Li J., Li Y., Wang G., Zhang L., Li T, & Li C. (2022). Vaccination of cats with Sad23L-nCoV-S vaccine candidate against major variants of SARS-CoV-2. Molecular Therapy. Methods & Clinical Development, 26, 181-190.
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4

SARS-CoV-2 Spike Protein-Specific T Cell Assay

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Mouse IFN-gamma ELISpot PLUS kits (MabTech) were used to determine SARS-CoV-2 S antigen-specific T lymphocyte response. Mouse splenocytes (5 × 105 cells/well) were stimulated with S peptides (5 μg/mL) in triplicates. Fifty-four peptides encoding amino acid sequence of SARS-CoV-2 S protein were predicted (http://www.iedb.org/) and synthesized by Guangzhou IGE Biotechnology LTD (11 (link)). Spots were counted with a CTL Immunospot Reader (Cellular Technology Ltd.). The results were expressed as spot forming cells (SFCs) per million cells.
Zou P., Zhang P., Deng Q., Wang C., Luo S., Zhang L., Li C, & Li T. (2023). Two Novel Adenovirus Vectors Mediated Differential Antibody Responses via Interferon-α and Natural Killer Cells. Microbiology Spectrum, 11(4), e00880-23.
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5

Tumor-specific IFN-γ Immune Response Quantification

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Tumor-specific immune responses to IFN-γ were measured in vitro by ELISpot assay. A Millipore Multiscreen HTS-IP plate was sterilized with 70% ethanol and coated with anti-mouse IFN-γ capture antibody overnight at 4 °C. Single-cell suspensions of splenocytes were obtained from CT26 tumor-carrying mice and seeded onto the plate at a concentration of 2 × 105 cells per well. Cells were stimulated with AH1 antigen (SPSYVYHQF, AS-64798; tebu-bio) for 42 h at 37 °C and then discarded. The plate was then incubated with biotin-conjugated anti-IFN-γ detection antibody at room temperature for 2 h, followed by incubation with streptavidin-HRP at r.t. for 2 h. AEC substrate solution (Sigma, Cat. AEC101, USA) was added for cytokine spot detection. Spot forming cells were imaged and counted with a CTL Immunospot Reader (Cellular Technology Ltd, USA).
Li Y., Ni K., Chan C., Guo N., Luo T., Han W., Culbert A., Weichselbaum R.R, & Lin W. (2021). Dimethylaminomicheliolide Sensitizes Cancer Cells to Radiotherapy for Synergistic Combination with Immune Checkpoint Blockade. Advanced therapeutics, 5(1), 2100160.
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6

Quantification of IFNγ-Producing Splenocytes

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The spleen was removed aseptically, pressed through a 40-µm cell strainer (Falcon), treated with Red Blood Cell lysis buffer (Sigma-Aldrich) to lyse erythrocytes, and washed with RPMI Medium 1640 supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 0.1 mg/ml streptomycin (all Thermo Fisher Scientific). Ethanol-activated MultiScreenHTS plates (Merck) were coated with purified rat anti-mouse IFNɣ antibody (clone R4-6A2, BD Bioscience) in PBS and blocked with RPMI Medium 1640 containing 10% FBS, 100 U/ml penicillin, and 0.1 mg/ml streptomycin. Splenocytes were seeded at 1 × 106 cells/well and stimulated with 0.1 µg/ml peptide. Concanavalin A (Thermo Fisher Scientific) at the same concentration was used as an assay high control for IFNɣ production. After 20 h of incubation, IFNɣ-producing cells were detected with a biotinylated rat anti-mouse IFNɣ antibody (clone XMG1.2, BD Bioscience), AKP Streptavidin (BD Bioscience), and BCTP/NBT as a substrate (Thermo Fisher Scientific) for color development. The CTL ImmunoSpot Reader (Cellular Technology Ltd) was used for quantification of spots.
Ballhausen A., Przybilla M.J., Jendrusch M., Haupt S., Pfaffendorf E., Seidler F., Witt J., Hernandez Sanchez A., Urban K., Draxlbauer M., Krausert S., Ahadova A., Kalteis M.S., Pfuderer P.L., Heid D., Stichel D., Gebert J., Bonsack M., Schott S., Bläker H., Seppälä T., Mecklin J.P., Ten Broeke S., Nielsen M., Heuveline V., Krzykalla J., Benner A., Riemer A.B., von Knebel Doeberitz M, & Kloor M. (2020). The shared frameshift mutation landscape of microsatellite-unstable cancers suggests immunoediting during tumor evolution. Nature Communications, 11, 4740.
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7

Mouse Splenic Cell Cytokine Secretion Assay

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Mouse splenic mononuclear cells were isolated using a Lymphoprep Kit (Solarbio, Beijing, China). Mouse IFN-γ ELISpotPLUS Kits and Mouse IL-4 ELISpotPLUS Kits (Mabtech Inc., Nacka Strand, Sweden) were used in accordance with the manufacturer’s recommended protocol. IFN-γ- and IL-4-secreting cells stimulated with the purified HBs (s28–39) polypeptide (IPQSLDSWWTSL, 2 μg/0.1 mL per well; Sangon Biotech Co., Ltd., Shanghai, China)35 (link) or with the purified Pn33Fps (5 μg/0.1 mL per well) were imaged and counted using a C.T.L. Immunospot reader (Cellular Technology Limited, OH, USA).
Qian W., Huang Z., Chen Y., Yang J., Wang L., Wu K., Chen M., Chen N., Duan Y., Shi J., Zhang Y, & Li Q. (2020). Elicitation of integrated immunity in mice by a novel pneumococcal polysaccharide vaccine conjugated with HBV surface antigen. Scientific Reports, 10, 6470.
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8

IFN-γ ELISPOT Assay for Mouse T Cells

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B6 and B6.E+ splenic CD4+ T cells were obtained by incubating cells with anti-CD4 microbeads for 30 min. at 4°C and flowing samples through magnetized LS positive selection columns (Miltenyi Biotec). 1.5 × 106 T cells were cultured for 48 h with equal numbers of irradiated Balb/c or SJL splenocytes, 20ng/mL phorbol 12-myristate 13-acetate (PMA) + 1μg/mL ionomycin, or medium only in 96-well MultiScreen-IP plates (Millipore) coated with purified anti-IFNγ (RA-6A2; eBioscience). Plates were then incubated with biotinylated anti-IFNγ (XMG1.2; eBioscience) and streptavidin-conjugated HRP (Southern Biotech), developed with 5-bromo-4-chloro-3-indoyl phosphate/nitro blue tetrazolium (BCIP/NBT; Sigma-Aldrich), read on a CTL ImmunoSpot reader, and analyzed using CTL ImmunoSpot 4.0 (Cellular Technology Limited).
Ni P.P., Wang Y, & Allen P.M. (2014). Both Positive and Negative Effects on Immune Responses by Expression of a Second Class II MHC Molecule. Molecular immunology, 62(1), 199-208.
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9

ELISpot Assay for SARS-CoV-2 RBD Immune Response

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ELISpot assays were used to evaluate cellular immune responses through measuring expression of IFN-γ, IL-2, IL-5, and GrzB by PBMC stimulated with RBD according to the manufacturer’s standard protocol (Cellular Technology Limited, Ohio, USA). Plates precoated with specific antibodies were washed with phosphate-buffered saline (PBS) and seeded with unfractionated PBMC at 250,000 cells/well. The wells were plated with unfractionated PBMC at 300,000 cells/well, and the cells were cultured with SARS-CoV-2 RBD at a concentration of 0.2 μg/mL. After incubation at 37°C for 24 h, the cells were removed and the plates processed according to the instructions of the manufacturer. The number of spots was determined automatically with an automatic CTL Immunospot reader (Cellular Technology, Shaker Heights, Ohio). The background was defined as the spots produced in the presence of antigen on day 0 before vaccination. All measurements were subtracted by the background values individually, while the subtracted values were corrected to 0. The results are expressed as the number of SFCs per 1,000,000 cells.
Zhao W., Chen W., Li J., Chen M., Li Q., Lv M., Zhou S., Bai S., Wang Y., Zhang L., Zhang P., Wang J., Zheng Q, & Wu J. (2022). Status of Humoral and Cellular Immune Responses within 12 Months following CoronaVac Vaccination against COVID-19. mBio, 13(3), e00181-22.
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10

IFN-gamma ELISpot Assay for T Cell Response

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Human (or mouse) IFN-gamma ELISpotPLUS kits (MabTech, Sweden) were used to determine antigen-specific T lymphocyte response. Marmoset PBMCs (2×105 cells/well) or mouse splenocytes (5×105 cells/well) were stimulated with peptides (10μg/ml M or E protein; Sino Biological, Beijing, China). Spots were counted with a CTL Immunospot Reader (Cellular Technology Ltd, Cleveland, USA). The result was presented with spot forming cells (SFCs) per million PBMCs or splenocytes.
Luo S., Zhao W., Ma X., Zhang P., Liu B., Zhang L., Wang W., Wang Y., Fu Y., Allain J.P., Li T, & Li C. (2020). A high infectious simian adenovirus type 23 vector based vaccine efficiently protects common marmosets against Zika virus infection. PLoS Neglected Tropical Diseases, 14(2), e0008027.
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