Ninety-six-well multiScreen filter plates (Millipore, USA) were pre-wetted with 35% ethanol then washed twice with PBS before coating. Plates were coated at 4°C overnight with 10µg/ml PR8 for B-cell ELISPOTs or 5µg/ml anti-mouse IL-2, IL-4, or IFN-γ (all from BD Biosciences) or IL-17 mAb (BioLegend, San Diego) for T-cell ELISPOTs. The plates were washed with PBS, then blocked with RPMI 1640+10% FCS. A total of 4×105 splenocytes and 5µg/ml PR8 were added in triplicates before the plates were incubated at 37°C for 24 hr (B-cell ELISPOT) or 48 hr (T-cell ELISPOT). The plates were washed 3 times with PBS-Tween 20 and 2µg/ml detection antibodies in PBS+10% FCS were added and incubate for overnight at 4°C. After discarding detection antibodies, plates were washed 3 times with PBS/Tween 20 and incubated with 1:200 dilution of HRP-Streptavidin (BD Biosciences) for 1 hr at RT. After washing spots were visualized using AEC substrate (BD Biosciences). The plates were washed twice with water, dried, and analyzed by Immunospot ELISPOT system (Cellular Technology, Shaker Heights, OH, USA).
Anti mouse il 2
Manufactured by BD
Sourced in United States
Anti-mouse IL-2 is a laboratory reagent used for the detection and quantification of mouse interleukin-2 (IL-2) in various biological samples. It is a specific antibody that binds to the IL-2 protein, allowing for its measurement and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Multiscreen filter plate, by Merck Group (2 mentions)
Il 17 mab, by BioLegend (2 mentions)
Streptavidin hrp, by BD (2 mentions)
Aec substrate, by BD (2 mentions)
Immunospot elispot system, by Cellular Technology (2 mentions)
Ifn γ, by BD (2 mentions)
Anti mouse ifn γ, by BioLegend (2 mentions)
Anti mouse cd8, by BioLegend (2 mentions)
Rabbit anti mouse hif1α antibody, by Novus Biologicals (2 mentions)
Mouse anti mouse tfam, by Santa Cruz Biotechnology (2 mentions)
Anti mouse tcf7 tcf1 antibody, by R&D Systems (2 mentions)
Anti mouse cd45, by BD (2 mentions)
Alexa fluor conjugated secondary antibody, by Thermo Fisher Scientific (2 mentions)
Cytofix cytoperm kit, by BD (2 mentions)
Foxp3 transcription factor staining buffer set, by Thermo Fisher Scientific (2 mentions)
Lsr 2, by BD (2 mentions)
Facscanto 2, by BD (2 mentions)
Anti mouse ifn γ, by BD (2 mentions)
Tnf α, by BD (1 mentions)
Immunospotseries 4, by Cellular Technology (1 mentions)
9 protocols using anti mouse il 2
1
ELISPOT Assay for B and T Cells
2
Quantification of Effector Memory CD8+ T Cells
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Surface stained Ova-specific effector memory CD8+ T cells were fixed and permeabilized with Cytofix/Cytoperm kit (BD Biosciences), followed by staining with rabbit anti-mouse HIF1α antibody (Novus Biologicals#nb100-449) or mouse anti-mouse TFAM (Santa Cruz Biotechnology#sc-166965) at 4°C for 1 hour. This was followed by staining with Alexa Fluor conjugated secondary antibodies (Molecular Probes) at 4°C for 1 hour. In case of intracellular staining of IL-2 and IFN-γ, splenocytes harvested from CD45.1+ mice were pre-treated with FcRγII/III (Fc blocker) and IgG2b anti-mouse CD16/CD32 antibodies, then stained with anti-mouse CD8 (Biolegend#100725), anti-mouse CD45.2 (BD PharMingen#561096) and anti-mouse Ova_tetramer before intracellular staining. Surface stained splenocytes were then fixed and permeabilized with Cytofix/Cytoperm kit (BD Biosciences#554714), followed by staining with anti-mouse IL-2 (BD PharMingen#554428) or anti-mouse IFN-γ (Biolegend# 505806). In case of intranuclear staining for TCF7, surface stained cells were fixed and permeabilized using Foxp3/Transcription factor staining buffer set (eBioscience#00-5523-00), followed by staining with anti-mouse TCF7/TCF1 antibody (R&D systems#FAB8224R). Stained cells were analyzed on BD FACSCantoII or BD LSRII.
3
Intracellular Cytokine Profiling Protocol
4
Cytokine Secretion Assay for T Cell Response
5
ELISPOT Assay for B-cell and T-cell Responses
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Ninety-six-well multiScreen filter plates (Millipore, USA) were pre-wetted with 35% ethanol then washed twice with PBS before coating. Plates were coated at 4 °C overnight with 10 μg/ml PR8 for B-cell ELISPOTs or 5 μg/ml anti-mouse IL-2, IL-4, or IFN-γ (all from BD Biosciences) or IL-17 mAb (BioLegend, San Diego) for T-cell ELISPOTs. The plates were washed with PBS, then blocked with RPMI 1640 + 10% FCS. A total of 4 × 105 splenocytes and 5 μg/ml PR8 were added in triplicates before the plates were incubated at 37 °C for 24 h (B-cell ELISPOT) or 48 h (T-cell ELISPOT). The plates were washed 3 times with PBS-Tween 20 and 2 μg/ml detection antibodies in PBS + 10% FCS were added and incubate for overnight at 4 °C. After discarding detection antibodies, plates were washed 3 times with PBS/Tween 20 and incubated with 1:200 dilution of HRP-Streptavidin (BD Biosciences) for 1 h at RT. After washing spots were visualized using AEC substrate (BD Biosciences). The plates were washed twice with water, dried, and analyzed by Immunospot ELISPOT system (Cellular Technology, Shaker Heights, OH, USA).
6
CD4+ Naïve T Cell Culture Protocol
7
Activate CD4+ Naive T-Cells in Vitro
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RPMI 1640 medium containing 10% FCS was used for CD4 naive T-cell culture. Antibodies used for cell culture were anti-mouse CD3 (145-2C11) and anti-mouse IL2 (300 u/ml) (BD BioSciences, San Jose, CA, USA).
8
Quantification of Effector Memory CD8+ T Cells
9
Cytokine Profiling of Mouse Splenic CD4 T Cells
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Primary mouse splenic CD4 T cell culture supernatants were collected at designated time points and analyzed for cytokine secretion. 96-well Maxisorp plates were coated overnight at 4°C with the appropriate capture antibody (anti-mouse IFNγ, anti-mouse IL-2, anti-IL-4, anti-IL-17A, and anti-IL-10; BD Biosciences). Non-specific protein binding was prevented by blocking wells with 5% BSA in PBS for 3 h at room temperature. Culture supernatants and standards (recombinant IFNγ and IL-2, carrier-free) were diluted appropriately and added to wells. The plate was incubated overnight at 4°C, with continuous rocking. Biotinylated detection antibodies were added to wells followed by TMB substrate reagents (BD Biosciences) at a 1:1 ratio. Color development was monitored, and the reaction was terminated by the addition of stop solution (2N H2SO4). Absorbance was read at 450 nm using a microplate reader. Cytokine concentrations were determined relative to the standard curves generated.
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