Msips4w10

Manufactured by Merck Group

Sourced in United States

The MSIPS4W10 is a laboratory equipment product from Merck Group. It is a specialized device designed for specific laboratory applications. Due to the technical nature of the product, a detailed description cannot be provided in a concise, unbiased, and factual manner without the risk of extrapolation or interpretation. Therefore, a comprehensive description for this product is not available at this time.

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Lab products found in correlation

Bcip nbt plus, by Mabtech (2 mentions) Staphylococcal enterotoxin b, by Merck Group (2 mentions) X vivo 20, by Lonza (2 mentions) Anti human ifn γ 1 d1k, by Mabtech (2 mentions) Bcip nbt, by Merck Group (2 mentions) X vivo 20 media, by Lonza (1 mentions) Aec substrate kit, by BD (1 mentions) Human albumin, by Merck Group (1 mentions) Immunospot analyzer, by Cellular Technology (1 mentions) 3 amino 9 ethylcarbazole aec substrate, by BD (1 mentions) 5 bromo 4 chloro 3 indolyl phosphate bcip nitro blue tetrazolium substrate solution, by Merck Group (1 mentions) Streptomycin, by Thermo Fisher Scientific (1 mentions) Penicillin, by Thermo Fisher Scientific (1 mentions) Fetal calf serum (fcs), by Thermo Fisher Scientific (1 mentions) Hepes, by Thermo Fisher Scientific (1 mentions) β mercaptoethanol, by Thermo Fisher Scientific (1 mentions) Streptomycin d penicillin, by Thermo Fisher Scientific (1 mentions) Immunospot software, by Cellular Technology (1 mentions) Extravidin phosphatase, by Merck Group (1 mentions) Streptavidin alkaline phosphatase, by Merck Group (1 mentions)

12 protocols using msips4w10

IFNγ ELISpot Assay for T Cell Detection

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T cell detection by IFNγ ELISpot assay was performed according to the manufacturer’s instructions (3321–2 A, Mabtech). Briefly, ELISpot PVDF plates (MSIPS4W10, Millipore) were pre-activated with 35% ethanol, coated with IFNγ-coating antibody and incubated overnight at 4–8 °C. Splenocytes (2 × 10⁵ if peptide-stimulated, 5 × 10⁴ if PMA/Ionomycin-stimulated) were added to duplicate wells and incubated overnight with 2 μg/mL peptides. Stimuli used were IE1, IE2 and pp65 peptide libraries as well as HLA-A2-restricted pp65- and IE1-specific immunodominant epitope peptides pp65 495–503 (NLVPMVATV) and IE1 316–324 (VLEETSVML) and HLA-B7-restricted pp65-specific immunodominant epitope peptide pp65 265-275 (RPHERNGFTVL). After 16–24 h, cells were removed from the wells, and IFNγ-detection antibody followed by streptavidin-ALP were added. Spots were developed using BCIP/NBT-plus (3650-10, Mabtech) and analyzed using CTL Analyzer immunoSpot plate reader. DMSO was used as negative control for splenocyte stimulation, and all values shown in Fig. 6 were DMSO-normalized.

Yll-Pico M., Park Y., Martinez J., Iniguez A., Kha M., Kim T., Medrano L., Nguyen V.H., Kaltcheva T., Dempsey S., Chiuppesi F., Wussow F, & Diamond D.J. (2024). Highly stable and immunogenic CMV T cell vaccine candidate developed using a synthetic MVA platform. NPJ Vaccines, 9, 68.

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Detecting NP-Specific Antibodies in Immunized Mice

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To detect the NP specific PCs in the spleens of NP-CGG immunized mice, 96 well sterile filter plates (MSIPS4W10- Millipore, Billerica, MA, USA) were coated with 10 μg/ml NP-BSA in sterile PBS over night at 4 °C. To discriminate the affinity of interactions, NP:BSA ratios of 4 (high affinity) and 30 (mixed affinity) were used. Plates were washed three times with PBS and then blocked with sterile PBS supplemented with 1% BSA for 2 hours at RT Followed by another three washes with sterile PBS. Cells harvested from the spleens of mice on day 14 post immunization were resuspended at three different dilutions in serum free X-VIVO™ 20 media (Lonza) supplemented with β-ME and plated into the ELISPOT plate. Plates were incubated at 37 °C, in a 5% CO₂ incubator, avoiding any vibrations for 20-24 hours. Cells were then discarded and plates were washed three times with PBS followed by another three washes with PBS-Tween 20 (0.5%). HRP-conjugated anti mouse IgG in PBS-Tween 20 (0.5%)-BSA (1%) solution was added to the plates. Following overnight incubation at 4°C, Plates were washed three times with PBS-Tween 20 (0.5%) followed by another three washes with PBS alone. Plates were developed using an AEC substrate kit (BD Biosciences) according to manufacturer’s guidelines. Evaluation and imaging of the developed ELISPOT plates were carried out by Zellnet Consulting, Fort Lee, NJ, USA.

Akkaya M., Akkaya B., Sheehan P.W., Miozzo P., Pena M., Qi C.F., Manzella-Lapeira J., Bolland S, & Pierce S.K. (2017). Immunization with a T cell-dependent protein antigen adjuvanted with DOTAP-CpG-B but not DOTAP-CpG-A induces robust germinal center responses and high affinity antibodies. European journal of immunology, 47(11), 1890-1899.

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Quantification of IFN-γ Secreting Cells

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White-bottom ELISpot HTS plates (MSIPS4W10, Millipore) were hydrophilized with 35% EtOH and subsequently coated with anti-human IFN-γ (1-D1K, Mabtech). Blocking was performed with X-Vivo-20 (Lonza) supplemented with 1% human albumin (Sigma-Aldrich). PBMCs were thawed and rested overnight in X-Vivo-20. Per well, 4 × 10⁵ cells were seeded and stimulated with 2 μg of peptide in a total volume of 100 μl. H3K27M (p14–40), H3wt (p14–40), or MOG (p35–55) was used as a stimulant, and 10% DMSO diluted in aqua ad iniectabilia (Braun) at equal volume was added as negative control. Positive controls were 1-μg staphylococcal enterotoxin B (Sigma-Aldrich), 0.05-μg CMV with 0.05-μg AdV per well, and CEFX Ultra SuperStim Pools of mixed, known peptide epitopes for MHC I– and MHC II–specific stimulation (PM-CEFX-4 and PM-CEFX-3, respectively; JPT Peptide Technologies). After 44 hours, detection of IFN-γ–secreting cells was performed by adding biotinylated anti-human IFN-γ antibodies (7-B6–1), streptavidin-ALP (both Mabtech), and ALP color development buffer (Bio-Rad). IFN-γ Spots were quantified by an ImmunoSpot Analyzer (ImmunoSpot/CTL Europe).

Boschert T., Kromer K., Lerner T., Lindner K., Haltenhof G., Tan C.L., Jähne K., Poschke I., Bunse L., Eisele P., Grassl N., Mildenberger I., Sahm K., Platten M., Lindner J.M, & Green E.W. (2024). H3K27M neoepitope vaccination in diffuse midline glioma induces B and T cell responses across diverse HLA loci of a recovered patient. Science Advances, 10(5), eadi9091.

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IFN-γ ELISpot Assay for Antigen-Specific T Cells

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The 96-well ELISpot plates (MSIPS4W10, Millipore) were prewetted with 70% EtOH and then precoated with anti-mouse IFN-γ capture antibody R46A2 (15 ug/ml, 50 ul/well) overnight at 4. The second day the plates were washed with PBS for twice and then blocked with 1% BSA in PBS. Splenocytes or cells from draining lymph nodes were obtained from the immunized B6N mice and added to the plates (1x10⁶ cells per well for splenocytes, 0.5x10⁶ cells per well for lymph node cells). The cells were stimulated with 50 ug/ml different peptides (peptides from human COMP library; P9; P9-1, P9-2, P9-3, P9-4) in triplicates at 37for 24 h. Concanavalin A (Con A, 1 ug/ml) used as a positive control. The plates were washed with PBS twice and a washing buffer (PBS containing 0.01% Tween 20) for 4 times followed by incubation with 50ul/well of biotinylated anti-IFN-γ (clone An18, 4 ug/ml in PBS/BSA 0.5%) at room temperature for 2 h. After washing the plates with the washing buffer for 5 times, streptavidin-alkaline phosphatase (50 ul/well, 1:2500 in PBS) was added to each well and incubated for 45 min at room temperature. After washing with the washing buffer for 3 times and PBS for 3 times, cytokine spots were visualized using Sigma Fast BCIP/nitroblue tetrazolium (100 ul/well) and enumerated using an ImmunoScan ELISpot Analyzer (CTL Europe).

Zhao Y., Urbonaviciute V., Xu B., Cai W., Sener Z., Ge C, & Holmdahl R. (2021). Cartilage Oligomeric Matrix Protein Induced Arthritis—A New Model for Rheumatoid Arthritis in the C57BL/6 Mouse. Frontiers in Immunology, 12, 631249.

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T-cell IFNγ ELISpot Assay Protocol

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T-cell detection by IFNγ ELISpot assay was performed according to the manufacturer’s instructions (3321-2A, Mabtech). ELISpot PVDF plates (MSIPS4W10, Millipore) were pre-activated with ethanol and coated with IFNγ-coating antibody. Splenocytes (2 × 10⁵ peptide-stimulated, 2 × 10⁴ PMA/Ionomycin-stimulated) were added to duplicate wells and incubated overnight with 2 µg/mL peptides. Stimuli included S and N peptide libraries; S1 subunit peptide pools covering peptides 1–86 (pool 1S1) and 87–168 (pool 2S1) of the S library; S2 subunit peptide pool that included peptides 169–316 of the S library; and peptide N26 (MKDLSPRWYFYYLGT) of the N peptide library. After 24 h, cells were removed, and IFNγ-detection antibody followed by streptavidin-ALP were added. Spots were developed using BCIP/NBT-plus (3650-10, Mabtech) and analyzed using AID ELISpot reader with AID ELISpot 5.0 iSpot software.

Chiuppesi F., Salazar M.D., Contreras H., Nguyen V.H., Martinez J., Park Y., Nguyen J., Kha M., Iniguez A., Zhou Q., Kaltcheva T., Levytskyy R., Ebelt N.D., Kang T.H., Wu X., Rogers T.F., Manuel E.R., Shostak Y., Diamond D.J, & Wussow F. (2020). Development of a multi-antigenic SARS-CoV-2 vaccine candidate using a synthetic poxvirus platform. Nature Communications, 11, 6121.

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ELISpot Assay for Detecting T Cell Responses

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ELISpot white-bottom multiscreen HTS plates (MSIPS4W10, Millipore) were coated with anti-human IFNγ (1-D1K, Mabtech) and blocked with X-Vivo-20 (Lonza) containing 2% human albumin (HA). PBMCs were thawed, rested overnight in X-Vivo medium and seeded at 4 × 10⁵ cells per well and stimulated with 2 μg peptides per well in 100 μl volume. PBMCs were stimulated with IDH1(R132H) (p123–142), wild-type IDH1 (p123–142), or MOG (p35–55) at equal concentrations or with peptide diluent aqua ad iniectabilia (Braun) with 10% DMSO (vehicle) at equal volume as negative controls, or with 1 μg staphylococcal enterotoxin B (Sigma-Aldrich) per well and 0.05 μg CMV with 0.05 μg AdV per well (both in 100 μl volume) as positive controls. After 40 h, IFNγ-producing cells were detected with biotinylated anti-human IFNγ antibodies (7-B6-1), streptavidin-ALP (both Mabtech) and ALP colour development buffer (Bio-Rad) and quantified using an ImmunoSpot Analyzer (Cellular Technology Ltd). Quality control was performed and reviewed by a second person. For categorization of T cell responses, transient T cell responses were defined as a spot count above 50 followed by a spot count of less than 50 at EOS. Sustained T cell responses were defined as a spot count above 50 followed by a spot count of more than 50 at EOS.

Platten M., Bunse L., Wick A., Bunse T., Le Cornet L., Harting I., Sahm F., Sanghvi K., Tan C.L., Poschke I., Green E., Justesen S., Behrens G.A., Breckwoldt M.O., Freitag A., Rother L.M., Schmitt A., Schnell O., Hense J., Misch M., Krex D., Stevanovic S., Tabatabai G., Steinbach J.P., Bendszus M., von Deimling A., Schmitt M, & Wick W. (2021). A vaccine targeting mutant IDH1 in newly diagnosed glioma. Nature, 592(7854), 463-468.

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ELISPOT Assay for Antibody-Secreting Cells

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Purified splenocytes or bone marrow (BM) cells were added at different dilutions to a 96-well 0.45 μm polyvinylidene difluoride membrane (cat. #MSIPS4W10; Millipore) previously coated overnight at 4°C with 2 μg/ml NP₂₀BSA and blocked with complete RPMI cell culture media for 2 h at 37°C. Plates with cells were incubated in a humid chamber 12 h at 37°C, 5% CO₂, then washed six times with PBS 0.01% Tween-20, followed by incubation with goat anti-mouse IgG1-HRP (A10551, 1/2,000; Life Technologies) diluted in culture media for 2 h at room temperature. Plates were washed and AEC substrate (3′ amino-9-ethylcarbazole; BD Bioscience) was added to reveal the spots. Images were acquired in an Axiophot MZ12 microscope and scored spots were counted from appropriate cell dilutions (2 × 10⁶ cells after primary immunization and 0.5 × 10⁶ cells for recall).

Litzler L.C., Zahn A., Dionne K.L., Sprumont A., Ferreira S.R., Slattery M.R., Methot S.P., Patenaude A.M., Hébert S., Kabir N., Subramani P.G., Jung S., Richard S., Kleinman C.L, & Di Noia J.M. (2023). Protein arginine methyltransferase 1 regulates B cell fate after positive selection in the germinal center in mice. The Journal of Experimental Medicine, 220(9), e20220381.

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Quantifying H1N1-Specific Antibody Responses

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H1N1-virus-specific antibody-secreting cells (ASCs) in the spleens of H1N1-challenged WT and db/db mice were identified by ELISPOT^{64 (link)}. Briefly, 96-well plates (Cat. # MSIPS4W10, Millipore) were pre-coated with purified H1N1 influenza virus A/PR/8/34 (5 mg/mL) at 4 °C overnight. Plates were washed and blocked with RPMI 1640 with 10% FBS at room temperature for 1 h. 1 × 10⁴ PI^-CD3^-B220⁺ cells from spleens of WT and db/db mice with H1N1 influenza virus on 9 days post-challenge were sorted and cultured for 12 h. Goat Anti-Mouse IgG Alkaline Phosphatase was then added after thoroughly wash and incubated for 1 h at room temperature. 5-Bromo-4-Chloro-3-Indolyl Phosphate (BCIP)/Nitro Blue Tetrazolium (NBT) substrate solution (Sigma–Aldrich) was added. Spots were monitored, stopped by gently washing, identified and counted by the Photoshop CS4 software (Adobe).

Deng J., Chen Q., Chen Z., Liang K., Gao X., Wang X., Makota F.V., Ong H.S., Wan Y., Luo K., Gong D., Yu X., Camuglia S., Zeng Q., Zhou T., Xue F., He J., Wei Y., Xiao F., Ma J., Hill D.L., Pierson W., Nguyen T.H., Zhou H., Wang Y., Shen W., Sun L., Li Z., Xia Q., Qian K., Ye L., Rockman S., Linterman M.A., Kedzierska K., Shen N., Lu L, & Yu D. (2021). The metabolic hormone leptin promotes the function of TFH cells and supports vaccine responses. Nature Communications, 12, 3073.

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Antigen-specific T-cell response analysis

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Single cell suspension from spleens of mice previously immunized for GPI arthritis was added to plates (MSIPS4W10, Millipore) previously coated with antibodies to IL-2 (clone JES6-IA12), IL-17(clone TC11-18H10) and a-IFNg(AN18) and stimulated for 48 h with 10 μM hGPI_325-339 in D-MEM supplemented with HEPES (GIBCO), streptomycin/D-penicillin (104 IU/ml penicillin, 10 mg/ml streptomycin; Invitrogen Life Technologies), b-mercaptoethanol (GIBCO), 10% fetal calf serum (GIBCO). Plates were washed and incubated with biotinylated antibodies to IL-2 (clone 5H4), IL-17 (clone TC11-8H4) and IFNγ (R46A2). Plates were washed and Extravidin-Phosphatase (Sigma) was added to wells and incubated for 45 min and washed. Plates were subsequently developed using BCIP/NBT(Sigma). Plates were scanned, analyzed and number of reactive cells were counted with ImmunoSpot software (Cellular Technology Ltd.).

Norin U., Rintisch C., Meng L., Forster F., Ekman D., Tuncel J., Klocke K., Bäcklund J., Yang M., Bonner M.Y., Lahore G.F., James J., Shchetynsky K., Bergquist M., Gjertsson I., Hubner N., Bäckdahl L, & Holmdahl R. (2021). Endophilin A2 deficiency protects rodents from autoimmune arthritis by modulating T cell activation. Nature Communications, 12, 610.

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Quantifying Antibody Responses via ELISA and ELISpot

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ELISA and ELISpot assays were performed according to standard protocols. Briefly, plates (ThermoFisher 442404) were coated with anti-mouse Ig(H+L) (Southern Biotech 1010-01) in a sodium carbonate/bicarbonate solution pH 9.6. Helicobacter ELISpot plates were coated with 200ng/well of heat-killed H. muridarum. Serum was serially diluted and detected using an HRP conjugated anti-mouse IgA antibody (Sigma A4789) and BD OptEIA TMB substrate (BD 555214). ELISpot plates (EMD Millipore MSIPS4W10) were coated the same as ELISA plates, but detection was performed with anti- IgM, IgA, IgG, κ, and λ antibodies conjugated to biotin (Southern Biotech). biotinylated antibodies were revealed with streptavidin-alkaline phosphatase (Sigma E2636) and developed with BCIP/NBT (Sigma B1911). ELISpot plates were imaged and spots were counted using a CTL Immunospot Analyzer (Cellular Technologies Limited).

Wilmore J.R., Gaudette B.T., Atria D.G., Hashemi T., Jones D.D., Gardner C.A., Cole S.D., Misic A.M., Beiting D.P, & Allman D. (2018). Commensal microbes induce serum IgA responses that protect against polymicrobial sepsis. Cell host & microbe, 23(3), 302-311.e3.

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